Inhibitors of Beta-Hydroxylase for Treatment of Cancer

ABSTRACT

The present invention relates to compounds which modulate (e.g., inhibit) the activity of beta-hydroxylase (e.g., Asparatyl (asparaginyl) β-hydroxylase (ASPH)), including novel 2-aryl-5-amino-3(2H)-furanone and 2-heteroaryl-5-amino-3(2H)-furanone compounds, pharmaceutical compositions thereof, methods for their synthesis, and methods of using these compounds to modulate the activity of ASPH in an a cell-free sample, a cell-based assay, and in a subject. Other aspects of the invention relate to use of the compounds disclosed herein to ameliorate or treat cell proliferation disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. §371 basedon International Application Number PCT/US2013/061050, filed Sep. 20,2013, which claims priority to, and the benefit of, U.S. provisionalapplication No. 61/704,014, filed Sep. 21, 2012, the entire contents ofwhich are incorporated herein by reference in their entirety.

INCORPORATION-BY-REFERENCE OF A SEQUENCE LISTING

The sequence listing contained in the file“761_(—)190_(—)010_US_ST25.txt”, created on Feb. 18, 2015 modified onFeb. 18, 2015 file size 19,903 bytes, is incorporated by reference inits entirety herein.

FIELD OF THE INVENTION

The invention relates to cell proliferation disorders, such as cancer.

BACKGROUND OF THE INVENTION

Hepatocellular carcinoma (HCC) is the fifth most prevalent, and thirdmost fatal type of cancer presently diagnosed in over a half-millionpeople, and which is on the increase globally (Fong et al. (1999) AnnSurg 229(6): 790-800). Local treatments, which include surgicalresection, liver transplantation and radiofrequency ablation, areconsidered as a first choice for the treatment of HCC. With improvementin these techniques, there has been progress on the early-stage therapyof HCC. Radiofrequency ablation has a demonstrated benefit forearly-stage disease, and it can be performed in patients with impairedliver function due to cirrhosis. However, many HCC tumors have highlymalignant phenotypes, which aggressively recur after local ablation evenif they were discovered at an early stage and have a very poorprognosis. Sorafenib is the only drug having a proven modest clinicalbenefit and approval as a systemic therapy for HCC. Therefore,development of a novel treatment approach for HCC and other Asparatyl(asparaginyl) β-hydroxylase (ASPH)-expressing solid tumors is urgentlyneeded.

SUMMARY OF THE INVENTION

Described herein is a family of compounds that inhibits β-hydroxylaseactivity of ASPH, which is highly overexpressed in cancers of the liver,pancreas, stomach, colon, breast, prostate, lung, brain as well as manyother tumor types. ASPH is necessary and sufficient to promote tumorcell migration, invasion, motility and distant metastatic spread both invitro and in vivo. Administration of these small molecule inhibitors ofβ-hydroxylase enzymatic activity reduce tumor development and growth aswell as distant metastatic spread to the liver and thus, useful as adrugs to treat a variety of deadly human tumors that overexpress ASPH.The invention encompasses compositions of matter of the small molecules,with and without pharmaceutically-acceptable excipients foradministration to human and animal subjects as well as the use of thesmall molecules in the treatment of human malignancies. The compoundsand methods prevent as well as slow the growth rate of establishedtumors and have low toxicity to normal cells.

In one aspect, this disclosure provides an ASPH inhibitory compound foruse in a method of reducing proliferation, migration, invasion, ormetastasis of a tumor cell in the treatment of cell proliferativedisorder, comprising contacting said tumor cell with the ASPH inhibitorycompound, wherein the ASPH inhibitory compound is of Formula Ia or Ib:

-   -   or a salt, ester, metabolite, prodrug, or solvate thereof,        wherein    -   Ar¹ is substituted or unsubstituted C₆-C₂₀ aryl or 5 to        20-membered heteroaryl;    -   X is C(O), C(S), or S(O)₂;    -   W¹ is a single bond, O, CR⁵⁰R⁵¹, or NR⁵² when X is CO and W¹ is        a single bond, CR⁵⁰R⁵¹, or NR⁵² when X is SO₂; and    -   each of R⁵⁰, R⁵¹, R⁵², and R⁵³ independently is selected from        the group consisting of hydrogen, substituted or unsubstituted        C₁-C₆ alkyl, substituted or unsubstituted C₂-C₆ alkenyl,        substituted or unsubstituted C₂-C₆ alkynyl, substituted or        unsubstituted C₆-C₂₀ aryl, substituted or unsubstituted C₇-C₂₆        arylalkyl, substituted or unsubstituted 5 to 20-membered        heteroaryl, and substituted or unsubstituted 6-26 membered        heteroarylalkyl.

In one embodiment, the compound for said use is of Formula Ia, or asalt, ester, metabolite, prodrug, or solvate thereof. The compound ofFormula Ia may have one or more of the following features whenapplicable.

For example, the compound is of Formula IIa:

-   -   or a salt, ester, metabolite, prodrug, or solvate thereof,        wherein    -   each of Ar¹ and Ar² independently is unsubstituted C₆-C₁₄ aryl,        unsubstituted 5 to 14-membered heteroaryl, or C₆-C₁₄ aryl or 5        to 14-membered heteroaryl each substituted with one or more        substituents selected from the group consisting of halo, CN,        NO₂, NO, N₃, OR_(a), NR_(a)R_(b), C(O)R_(a), C(O)OR_(a),        C(O)NR_(a)R_(b), NR_(b)C(O)R_(a), —S(O)_(b)R_(a),        —S(O)_(b)NR_(a)R_(b), or R_(S1), in which R_(S1) is C₁-C₆ alkyl,        C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃ ⁻C₈ cycloalkyl, C₆-C₁₀ aryl, 5-        or 6-membered heteroaryl, or 4 to 12-membered heterocycloalkyl,        b is 0, 1, or 2, each of R_(a) and R_(b), independently is H or        R_(S2), and R_(S2) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered        heterocycloalkyl, or 5- or 6-membered heteroaryl; and each of        R_(S1) and R_(S2), is optionally substituted with one or more        substituents selected from the group consisting of halo, OH,        oxo, C(O)OH, C(O)O—C₁-C₆ alkyl, CN, C₁-C₆ alkyl, C₁-C₆ alkoxyl,        amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈        cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, and        5- or 6-membered heteroaryl.

For example, R⁵³ is unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substitutedwith one or more substituents selected from halo, OH, CN, and amino.

For example, X is S(O)₂ and W¹ is CR⁵⁰R⁵¹.

For example, X is S(O)₂ and W¹ is a single bond.

For example, X is C(O) and W¹ is O, or X is C(S) and W¹ is NR⁵².

For example, each of R⁵⁰, R⁵¹, and R⁵² independently is H, unsubstitutedC₁-C₆ alkyl, or C₁-C₆ alkyl substituted with one or more substituentsselected from halo, OH, CN, and amino.

For example, each of Ar¹ and Ar² independently is phenyl, naphthyl, or 5to 10-membered heteroaryl, each of which is optionally substituted withone or more substituents selected from the group consisting of halo, CN,NO₂, NO, N₃, OR_(a), NR_(a)R_(b), C(O)R_(a), C(O)OR_(a),C(O)NR_(a)R_(b), NR_(b)C(O)R_(a), —S(O)_(b)R_(a), —S(O)_(b)NR_(a)R_(b),or R_(S1), in which R_(S1) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5- or 6-membered heteroaryl, or 4 to12-membered heterocycloalkyl, b is 0, 1, or 2, each of R_(a) and R_(b),independently is H or R_(S2), and R_(S2) is C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, or 5- or 6-membered heteroaryl; and each of R_(S1) andR_(S2), is optionally substituted with one or more substituents selectedfrom the group consisting of halo, OH, oxo, C(O)OH, C(O)O—C₁-C₆ alkyl,CN, C₁-C₆ alkyl, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl.

For example, each of Ar¹ and Ar² independently is phenyl, naphthyl, or 5to 10-membered heteroaryl, each of which is optionally substituted withone or more substituents selected from the group consisting of halo, CN,NO₂, NO, N₃, OR_(a), NR_(a)R_(b), C(O)R_(a), C(O)OR_(a), or R_(S1), inwhich R_(S1) is C₁-C₆ alkyl, each of R_(a) and R_(b), independently is Hor R_(S2), and R_(S2) is C₁-C₆ alkyl; and each of R_(S1) and R_(S2), isoptionally substituted with one or more substituents selected from thegroup consisting of halo, OH, C₁-C₆ alkoxyl, amino, mono-C₁-C₆alkylamino, and di-C₁-C₆ alkylamino.

For example, each of Ar¹ and Ar² independently is selected from phenyl,1-naphthyl, 2-naphthyl, 2-furanyl, 2-thiazolyl, 2-pyridyl, 3-pyridyl,4-pyridyl, 2-quinolinyl, 3-quinolinyl, 4-quinolinyl, 2-chlorophenyl,3-chlorophenyl, 4-chlorophenyl, 2-fluorophenyl, 3-fluorophenyl,4-fluorophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl,4-trifluoromethylphenyl, 2-cyanophenyl, 3-cyanophenyl, 4-cyanophenyl,3-carboxymethylphenyl, 2-methoxyphenyl, 3-methoxyphenyl,4-methoxyphenyl, 2,3-dichlorophenyl, 2,4-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl,3,4-difluorophenyl, 3,5-difluorophenyl, 2,3-dimethoxyphenyl,2,4-dimethoxyphenyl, 2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl,3,5-dimethoxyphenyl, 2-chloro-6-fluorophenyl, 3-chloro-4-fluorophenyl,2-chloro-4-fluorophenyl, 4-chloro-3-fluorophenyl,3-chloro-2-fluorophenyl, 2-chloro-5-fluorophenyl,4-chloro-2-fluorophenyl, and 5-chloro-2-fluorophenyl.

In another embodiment, the compound for said use is of Formula Ib, or asalt, ester, metabolite, prodrug, or solvate thereof. The compound ofFormula Ib may have one or more of the following features whenapplicable.

For example, R⁵³ is unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substitutedwith one or more substituents selected from halo, OH, CN, and amino.

For example, R⁵³ is unsubstituted methyl or ethyl.

For example, X is S(O)₂ and W¹ is CR⁵⁰R⁵¹.

For example, X is S(O)₂ and W¹ is a single bond.

For example, X is C(O) and W¹ is O, or X is C(S) and W¹ is NR⁵².

For example, each of R⁵⁰, R⁵¹, and R⁵² independently is H, unsubstitutedC₁-C₆ alkyl, or C₁-C₆ alkyl substituted with one or more substituentsselected from halo, OH, CN, and amino.

For example, Ar¹ is phenyl, naphthyl, or 5 to 10-membered heteroaryl,each of which is optionally substituted with one or more substituentsselected from the group consisting of halo, CN, NO₂, NO, N₃, OR_(a),NR_(a)R_(b), C(O)R_(a), C(O)OR_(a), C(O)NR_(a)R_(b), NR_(b)C(O)R_(a),—S(O)_(b)R_(a), —S(O)_(b)NR_(a)R_(b), or R_(S1), in which R_(S1) isC₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 5- or 6-membered heteroaryl, or 4 to 12-membered heterocycloalkyl,b is 0, 1, or 2, each of R_(a) and R_(b), independently is H or R_(S2),and R_(S2) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or6-membered heteroaryl; and each of R_(S1) and R_(S2), is optionallysubstituted with one or more substituents selected from the groupconsisting of halo, OH, oxo, C(O)OH, C(O)O—C₁-C₆ alkyl, CN, C₁-C₆ alkyl,C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, and 5- or6-membered heteroaryl.

For example, Ar¹ is phenyl, naphthyl, or 5 to 10-membered heteroaryl,each of which is optionally substituted with one or more substituentsselected from the group consisting of halo, CN, NO₂, NO, N₃, OR_(a),NR_(a)R_(b), C(O)R_(a), C(O)OR_(a), or R_(S1), in which R_(S1) is C₁-C₆alkyl, each of R_(a) and R_(b), independently is H or R_(S2), and R_(S2)is C₁-C₆ alkyl; and each of R_(S1) and R_(S2), is optionally substitutedwith one or more substituents selected from the group consisting ofhalo, OH, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, and di-C₁-C₆alkylamino.

For example, Ar¹ is selected from phenyl, 1-naphthyl, 2-naphthyl,2-furanyl, 2-thiazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-quinolinyl,3-quinolinyl, 4-quinolinyl, 2-chlorophenyl, 3-chlorophenyl,4-chlorophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl,2-trifluoromethylphenyl, 3-trifluoromethylphenyl,4-trifluoromethylphenyl, 2-cyanophenyl, 3-cyanophenyl, 4-cyanophenyl,3-carboxymethylphenyl, 2-methoxyphenyl, 3-methoxyphenyl,4-methoxyphenyl, 2,3-dichlorophenyl, 2,4-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl,3,4-difluorophenyl, 3,5-difluorophenyl, 2,3-dimethoxyphenyl,2,4-dimethoxyphenyl, 2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl,3,5-dimethoxyphenyl, 2-chloro-6-fluorophenyl, 3-chloro-4-fluorophenyl,2-chloro-4-fluorophenyl, 4-chloro-3-fluorophenyl,3-chloro-2-fluorophenyl, 2-chloro-5-fluorophenyl,4-chloro-2-fluorophenyl, and 5-chloro-2-fluorophenyl.

In one embodiment, said tumor cell expresses ASPH.

In certain embodiments, said cell proliferative disorder comprisesPancreatic Cancer, Hepatocellular Cancer, Cholangiocarcinoma, Lungcancer, Colon Cancer, Breast Cancer, Prostatic Cancer, and Glioblastoma.

In one embodiment, said compound is administered intravenously, orally,or subcutaneously.

In one embodiment, said compound is administered at a dose of 0.01 to 50milligrams/kilogram of body weight.

In another aspect, this disclosure features a compound of Formula Ia:

or a salt, ester, metabolite, prodrug, or solvate thereof, wherein

Ar¹ is substituted or unsubstituted C₆-C₂₀ aryl or 5 to 20-memberedheteroaryl;

X is C(O), C(S), or S(O)₂;

W¹ is a single bond, O, CR⁵⁰R⁵¹, or NR⁵² when X is CO and W¹ is a singlebond, CR⁵⁰R⁵¹, or NR⁵² when X is SO₂; and

each of R⁵⁰, R⁵¹, R⁵², and R⁵³ independently is selected from the groupconsisting of hydrogen, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted C₂-C₆ alkenyl, substituted or unsubstitutedC₂-C₆ alkynyl, substituted or unsubstituted C₆-C₂₀ aryl, substituted orunsubstituted C₇-C₂₆ arylalkyl, substituted or unsubstituted 5 to20-membered heteroaryl, and substituted or unsubstituted 6-26 memberedheteroarylalkyl, provided that when Ar¹ is 4-chlorophenyl, then R⁵³ isnot methyl or unsubstituted phenyl.

The compound of Formula Ia may have one or more of the followingfeatures when applicable.

For example, the compound is of Formula IIa:

or a salt, ester, metabolite, prodrug, or solvate thereof, wherein

each of Ar¹ and Ar² independently is unsubstituted C₆-C₁₄ aryl,unsubstituted 5 to 14-membered heteroaryl, or C₆-C₁₄ aryl or 5 to14-membered heteroaryl each substituted with one or more substituentsselected from the group consisting of halo, CN, NO₂, NO, N₃, OR_(a),NR_(a)R_(b), C(O)R_(a), C(O)OR_(a), C(O)NR_(a)R_(b), NR_(b)C(O)R_(a),—S(O)_(b)R_(a), —S(O)_(b)NR_(a)R_(b), or R_(S1), in which R_(S1) isC₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 5- or 6-membered heteroaryl, or 4 to 12-membered heterocycloalkyl,b is 0, 1, or 2, each of R_(a) and R_(b), independently is H or R_(S2),and R_(S2) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or6-membered heteroaryl; and each of R_(S1) and R_(S2), is optionallysubstituted with one or more substituents selected from the groupconsisting of halo, OH, oxo, C(O)OH, C(O)O—C₁-C₆ alkyl, CN, C₁-C₆ alkyl,C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, and 5- or6-membered heteroaryl.

For example, R⁵³ is unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substitutedwith one or more substituents selected from halo, OH, CN, and amino.

For example, X is S(O)₂ and W¹ is CR⁵⁰R⁵¹.

For example, X is S(O)₂ and W¹ is a single bond.

For example, X is C(O) and W¹ is O, or X is C(S) and W¹ is NR⁵².

For example, each of R⁵⁰, R⁵¹, and R⁵² independently is H, unsubstitutedC₁-C₆ alkyl, or C₁-C₆ alkyl substituted with one or more substituentsselected from halo, OH, CN, and amino.

For example, each of Ar¹ and Ar² independently is phenyl, naphthyl, or 5to 10-membered heteroaryl, each of which is optionally substituted withone or more substituents selected from the group consisting of halo, CN,NO₂, NO, N₃, OR_(a), NR_(a)R_(b), C(O)R_(a), C(O)OR_(a),C(O)NR_(a)R_(b), NR_(b)C(O)R_(a), —S(O)_(b)R_(a), —S(O)_(b)NR_(a)R_(b),or R_(S1), in which R_(S1) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5- or 6-membered heteroaryl, or 4 to12-membered heterocycloalkyl, b is 0, 1, or 2, each of R_(a) and R_(b),independently is H or R_(S2), and R_(S2) is C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, or 5- or 6-membered heteroaryl; and each of R_(S1) andR_(S2), is optionally substituted with one or more substituents selectedfrom the group consisting of halo, OH, oxo, C(O)OH, C(O)O—C₁-C₆ alkyl,CN, C₁-C₆ alkyl, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl.

For example, each of Ar¹ and Ar² independently is phenyl, naphthyl, or 5to 10-membered heteroaryl, each of which is optionally substituted withone or more substituents selected from the group consisting of halo, CN,NO₂, NO, N₃, OR_(a), NR_(a)R_(b), C(O)R_(a), C(O)OR_(a), or R_(S1), inwhich R_(S1) is C₁-C₆ alkyl, each of R_(a) and R_(b), independently is Hor R_(S2), and R_(S2) is C₁-C₆ alkyl; and each of R_(S1) and R_(S2), isoptionally substituted with one or more substituents selected from thegroup consisting of halo, OH, C₁-C₆ alkoxyl, amino, mono-C₁-C₆alkylamino, and di-C₁-C₆ alkylamino.

For example, each of Ar¹ and Ar² independently is selected from phenyl,1-naphthyl, 2-naphthyl, 2-furanyl, 2-thiazolyl, 2-pyridyl, 3-pyridyl,4-pyridyl, 2-quinolinyl, 3-quinolinyl, 4-quinolinyl, 2-chlorophenyl,3-chlorophenyl, 4-chlorophenyl, 2-fluorophenyl, 3-fluorophenyl,4-fluorophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl,4-trifluoromethylphenyl, 2-cyanophenyl, 3-cyanophenyl, 4-cyanophenyl,3-carboxymethylphenyl, 2-methoxyphenyl, 3-methoxyphenyl,4-methoxyphenyl, 2,3-dichlorophenyl, 2,4-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl,3,4-difluorophenyl, 3,5-difluorophenyl, 2,3-dimethoxyphenyl,2,4-dimethoxyphenyl, 2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl,3,5-dimethoxyphenyl, 2-chloro-6-fluorophenyl, 3-chloro-4-fluorophenyl,2-chloro-4-fluorophenyl, 4-chloro-3-fluorophenyl,3-chloro-2-fluorophenyl, 2-chloro-5-fluorophenyl,4-chloro-2-fluorophenyl, and 5-chloro-2-fluorophenyl.

This disclosure also provides a pharmaceutical composition comprising apharmaceutically acceptable carrier and a compound of Formula Ia or IIa,or a salt, ester, metabolite, prodrug, or solvate thereof.

In yet another aspect, the invention features a method of producing acompound of Formula IIa, wherein X is S(O)₂ and W¹ is CR⁵⁰R⁵¹. Themethod includes:

contacting an amine compound of the Formula (IIIa)

with a sulfonyl chloride of the Formula ClSO₂(CR⁵⁰R⁵¹)Ar² under asuitable condition to produce a compound of Formula IIa.

Also provided in the present disclosure is method for treating orpreventing a cell proliferative disorder, comprising administering to asubject in need thereof a therapeutically efficient amount of a compounddescribed herein, such as those of Formula Ia or Ib, or a salt, ester,metabolite, prodrug, or solvate thereof described herein. In oneembodiment, the compound is of Formula IIa or a salt, ester, metabolite,prodrug, or solvate thereof described herein. In another embodiment, thecompound is of Formula Ib or a salt, ester, metabolite, prodrug, orsolvate thereof described herein, wherein R⁵³ is unsubstituted C₁-C₆alkyl or C₁-C₆ alkyl substituted with one or more substituents selectedfrom halo, OH, CN, and amino.

In embodiments, the methods lead to at least a 10%, 20%, 50%, 2-fold,5-fold, 10-fold or more reduction in tumor mass, volume, or weightcompared to untreated tumors or untreated patients. In some cases, thetumor is completely eradicated. Similarly, the compounds inhibitmetastasis, e.g., by inhibiting tumor cell migration or invasion, e.g.,by at least 10%, 20%, 50%, 2-fold, 5-fold, 10-fold compared to untreatedtumors or tumor cell metastasis in untreated patients.

The small molecule inhibitors are administered to subjects in need oftreatment for a cell proliferative disease such as cancer in sufficientamounts of the compounds to reach blood concentrations varying between0.1 and 100 micro molar. The compound is administered using standardregimens, e.g., daily, every other day, every third day, every fifth dayor every seventh day. The compound is administered intravenously,orally, or subcutaneous. The amount given to reach the blood levelsdescribed above ranges from 0.01 to 50 milligrams/kilogram of bodyweight. The compositions and methods are suitable for treatment ofsubjects diagnosed as suffering from cancer, including those who haveundergone another form of treatment for cancer, e.g., otherchemotherapeutic agents, radiation, and surgery, and/or are diagnosedwith metastatic disease or are at risk of developing metastasis. Themammal can be any mammal, e.g., a human, a primate, a mouse, a rat, adog, a cat, a horse, as well as livestock or animals grown for foodconsumption, e.g., cattle, sheep, pigs, chickens, and goats. In apreferred embodiment, the mammal is a human.

Also described is use of an ASPH inhibitory compound of this inventionfor the manufacture of a medicament for the use in reduction ofproliferation, migration, invasion, or metastasis of a tumor cell in thetreatment of cancer.

In yet another aspect, the disclosure provides a method determining ASPHactivity by contacting an EGF-like domain peptide with adetectably-labeled α-ketoglutarate and ASPH enzyme and measuringβ-hydroxylase activity. In one embodiment, said α-ketoglutarate is¹⁴C-labelled, and wherein β-hydroxylase activity is measured bydetecting liberated ¹⁴CO₂. In one embodiment, said liberated ¹⁴CO₂ iscaptured on a filter and radioactivity quantified. In one embodiment,the method further includes contacting said ASPH enzyme with a candidatecompound, wherein a decrease in β-hydroxylase activity in the presenceof said compound compared to the absence of said compound indicates thatsaid compound inhibits ASPH enzyme activity. In one embodiment, saidEGF-like domain comprises the amino acid sequence of SEQ ID NO:1.

(SEQ ID NO: 1) DGDQCETSPCQNQGKCKDGLGEYTCTCLEGFEGKNCELF

In one embodiment, said EGF-like domain peptide comprises the consensussequence of SEQ ID NO:2.

(SEQ ID NO: 2) CDXXXCXXKXGNGXCDXXCNNAACXXDGXDC

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. All references cited herein are incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D are photomicrographs, and FIG. 1 E is a bar graph showingASPH expression in pancreatic cancer, normal pancreas and neuroendocrinepancreatic tumors. (A) represents ASPH expression in PC at 40× and (B)400× with mAb RC-50. (C) shows lack of ASPH expression in normalpancreas at 40× and (D) 400×. (E) shows ASPH immunoreactivity.

FIGS. 2A-D are photomicrographs showing ASPH expression in human HCCtumors by immunohistochemical staining (HIS) using RC-50 mAb. Note thatmost if not all tumor cells highly express the ASPH protein. (A) normalliver; (B, C and D) human HCC tumors.

FIG. 3A is a diagram of the biochemical reaction catalyzed by ASPHderived from PC cell lysates. FIG. 3B is a cartoon illustrating theread-out to quantify ASPH activity in PC cells and tumors. FIG. 3C is animage of membrane was obtained by a phosphor-imager after a 16 hourexposure of the trapping filter to a phospho-screen. FIG. 3D is a bargraph showing intensities were calculated by Image J software andcompared to a DMSO control. Vertical bars represent standard deviation.These figures show methods of measurement and data relating to ASPHcatalytic activity.

FIG. 4A is a line graph showing tumor volume, and FIG. 4B is a bar graphshowing tumor weight. The data show an anti-tumor effect of Compound 310{8c or MO-I-1100} and PC tumor growth initiated with the humanpancreatic HPAFII cell line. There were 15 nude mice in each group. i.p.injection (20 mg/kg/mouse). Vertical bars; standard error.

FIG. 5A-B are line graphs showing the effect of Compound 310 {8c orMO-I-1100} on metabolic activity (A), and proliferation (B). FIG. 5C isa bar graph showing effect of the inhibitor on viability (C). Note thatNIH 3T3 cells that lack ASPH expression were resistant to the antitumoractivity of Compound 310 {8c or MO-I-1100}.

FIG. 6A is a photograph, and FIG. 6B is a bar graph showing colonyformation (a test of malignant potential) produced by FOCUS HCC cellswas strikingly inhibited by treatment with 5 μM of Compound 310 {8c orMO-I-1100}.

FIG. 7A is an image of membrane was obtained by a phosphor-imager, andFIG. 7B is a bar graph showing that both Compound 405 {5c or MO-I-500}and Compound 310 {8c or MO-I-1100} inhibit ASPH enzymatic activity(β-hydroxylase) using the high through-put assay described in FIGS.3A-D.

FIGS. 8A-B are bar graphs, FIG. 8C is a photograph, and FIG. 8D is a bargraph showing the effect of Compound 405 {5c or MO-I-500} on cellmetabolism (A), proliferation (B), and colony formation (C, D). Notethat both FOCUS HCC cells (express ASPH) and NIH-3T3 cells (do notexpress ASPH) were growth inhibited indicating that Compound 405 {5c orMO-I-500} is not specific for ASPH although it strikingly inhibitscolony formation of FOCUS cells at 1.25 μM.

FIG. 9A is a photograph, and FIGS. 9B-C are bar graphs showing thatCompound 310 {8c or MO-I-1100} treatment of FOCUS HCC cells reduces thenumber (A, B) and size (A, C) of colony formation in soft agar thusreducing anchorage independent cell growth as a rigid test of malignanttransformation.

FIG. 10A is a photograph, and FIGS. 10B-C are bar graphs showing the invitro effects of Compound 405 {5c or MO-I-500} for anchorage independentcell growth. Note the striking reduction in the number and size of FOCUScell colonies following treatment with 0.5, 1, and 5 μM of Compound 405{5c or MO-I-500}.

FIGS. 11A-D are bar graphs showing the effect of Compound 310 {8c orMO-I-1100} on cell migration and invasion of FOCUS HCC cells. Note thatthis compound significantly inhibited the migratory (A), and invasion(D), properties of this human liver cancer cell line.

FIGS. 12A-B are bar graphs showing the in vitro effects of ASPHinhibitors Compound 405 {5c or MO-I-500} and Compound 310 {8c orMO-I-1100} on cell motility and invasiveness in a human HCC cell line(FOCUS).

FIGS. 13A is a photograph, and FIG. 13B is a line graph showing thatCompound 310 {8c or MO-I-1100} treatment strikingly reduces the size andgrowth of human liver cancer in vivo using an immunodeficient murinesubcutaneous xenograft model.

TERMS AND DEFINITIONS

The following is a list of abbreviations, plus terms and theirdefinitions, used throughout the specification and the claims:

General abbreviations and their corresponding meanings include: aa orAA=amino acid; mg=milligram(s); ml or mL=milliliter(s);mm=millimeter(s); mM=millimolar; nmol=nanomole(s); pmol=picomole(s);ppm=parts per million; RT=room temperature; U=units; ug, μg=microgram(s); ul, μl=micro liter(s); uM, μM=micromolar, TEA=triethylamine,LDA=lithium diisopropyl amine, THF=tetrahydrofuran,DMAP=4-dimethylaminopyridine, DMF=N,N′-dimethylformamide.

The terms “cell” and “cells”, which are meant to be inclusive, refer toone or more cells which can be in an isolated or cultured state, as in acell line comprising a homogeneous or heterogeneous population of cells,or in a tissue sample, or as part of an organism, such as an insectlarva or a transgenic mammal.

The term “amino acid” encompasses both naturally occurring andnon-naturally occurring amino acids unless otherwise designated.

The term “an effective amount” means an amount of the substance inquestion which produces a statistically significant effect. For example,an “effective amount” for therapeutic uses is the amount of thecomposition comprising an active compound herein required to provide aclinically significant alteration in a measurable trait. Such effectiveamounts will be determined using routine optimization techniques and aredependent on the particular condition to be treated, the condition ofthe patient, the route of administration, dosage required for thecompounds of the invention is manifested as that which induces astatistically significant difference between treatment and controlgroups.

A “therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of modulator mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the modulator to elicit adesired response in the individual. Dosage regimens may be adjusted toprovide the optimum therapeutic response. A therapeutically-effectiveamount is also one in which any toxic or detrimental effects of themodulator are outweighed by the therapeutically beneficial effects.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. A prophylactically effective amount can bedetermined as described above for the therapeutically-effective amount.Typically, since a prophylactic dose is used in subjects prior to or atan earlier stage of disease, the prophylactically effective amount willbe less than the therapeutically-effective amount.

As used herein, the term “cell proliferative disorder” refers toconditions in which unregulated or abnormal growth, or both, of cellscan lead to the development of an unwanted condition or disease, whichmay or may not be cancerous. Exemplary cell proliferative disorders thatmay be treated with the compounds of the invention encompass a varietyof conditions wherein cell division is deregulated. Exemplary cellproliferative disorder include, but are not limited to, neoplasms,benign tumors, malignant tumors, pre-cancerous conditions, in situtumors, encapsulated tumors, metastatic tumors, liquid tumors, solidtumors, immunological tumors, hematological tumors, cancers, carcinomas,leukemias, lymphomas, sarcomas, and rapidly dividing cells. The term“rapidly dividing cell” as used herein is defined as any cell thatdivides at a rate that exceeds or is greater than what is expected orobserved among neighboring or juxtaposed cells within the same tissue. Acell proliferative disorder includes a precancer or a precancerouscondition. A cell proliferative disorder includes cancer. The methodsand uses provided herein can be or may be used to treat or alleviate asymptom of cancer or to identify suitable candidates for such purposes.The term “cancer” includes solid tumors, as well as, hematologic tumorsand/or malignancies. A “precancer cell” or “precancerous cell” is a cellmanifesting a cell proliferative disorder that is a precancer or aprecancerous condition. A “cancer cell” or “cancerous cell” is a cellmanifesting a cell proliferative disorder that is a cancer. Anyreproducible means of measurement may be used to identify cancer cellsor precancerous cells. Cancer cells or precancerous cells can beidentified by histological typing or grading of a tissue sample (e.g., abiopsy sample). Cancer cells or precancerous cells can be identifiedthrough the use of appropriate molecular markers.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, to alleviate the symptoms or complicationsof a disease, condition or disorder, or to eliminate the disease,condition or disorder. The term “treat” can also include treatment of acell in vitro or an animal model.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, can or may alsobe used to prevent a relevant disease, condition or disorder, or used toidentify suitable candidates for such purposes. As used herein,“preventing,” “prevent,” or “protecting against” describes reducing oreliminating the onset of the symptoms or complications of such disease,condition or disorder.

As used herein, the term “alleviate” is meant to describe a process bywhich the severity of a sign or symptom of a disorder is decreased.Importantly, a sign or symptom can be alleviated without beingeliminated. The administration of pharmaceutical compositions of theinvention can or may lead to the elimination of a sign or symptom,however, elimination is not required. Effective dosages should beexpected to decrease the severity of a sign or symptom. For instance, asign or symptom of a disorder such as cancer, which can occur inmultiple locations, is alleviated if the severity of the cancer isdecreased within at least one of multiple locations.

As used herein, a “subject” is interchangeable with a “subject in needthereof”, both of which refer to a subject having a cell proliferationdisorder, or a subject having an increased risk of developing suchdisorder relative to the population at large. A “subject” includes amammal. The mammal can be e.g., a human or appropriate non-human mammal,such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep ora pig. The subject can also be a bird or fowl. In one embodiment, themammal is a human. A subject in need thereof can be one who has beenpreviously diagnosed or identified as having cancer or a precancerouscondition. A subject in need thereof can also be one who has (e.g., issuffering from) cancer or a precancerous condition. Alternatively, asubject in need thereof can be one who has an increased risk ofdeveloping such disorder relative to the population at large (i.e., asubject who is predisposed to developing such disorder relative to thepopulation at large). A subject in need thereof can have a precancerouscondition. The term “animal” includes human beings.

The term “optionally substituted” moiety refers to either unsubstitutedchemical moiety (e.g., alkyl, aryl, heteroaryl, etc.) or a chemicalmoiety having designated substituents replacing one or more hydrogenatoms on one or more carbons of the hydrocarbon backbone. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “substituted aryl or heteroaryl” refers to aromatic orheteroaromatic rings may contain one or more substituents such as —OH,SH, —CN, —F, —Cl, —Br, —R, —NO₂—NO, —NH2, —NHR, —NRR, —C(O)R, —C(O)OH,—C(O)OR, —C(O)NH2, —C(O)NHR, —C(O)NRR, and the like where each R isindependently (C₁-C₅) alkyl, substituted (C₁-C₆) alkyl, (C₂-C₆) alkenyl,substituted (C₂-C₆) alkenyl, (C₂-C₆) alkynyl, substituted (C₂-C₆)alkynyl, (C₅-C₂₀) aryl, substituted (C₅-C₂₀) aryl, (C₆-C₂₆) arylalkyl,substituted (C₆-C₂₆) arylalkyl, 5-20 membered heteroaryl, substituted5-20 membered heteroaryl, 6-26 membered heteroarylalkyl or substituted6-26 membered heteroarylalkyl.

An “arylalkyl” or an “aralkyl” moiety is an alkyl substituted with anaryl (e.g., phenylmethyl (benzyl)). An “alkylaryl” moiety is an arylsubstituted with an alkyl (e.g., methylphenyl).

A “derivative” of a compound X (e.g., a peptide or amino acid) refers toa form of X in which one or more reactive groups on the compound havebeen derivatized with a substituent group. Peptide derivatives includepep tides in which an amino acid side chain, the peptide backbone, orthe amino' or carboxy-terminus has been derivatized (e.g., peptidiccompounds with 5 methylated amide linkages).

An “analogue” of a compound X refers to a compound which retainschemical structures of X necessary for functional activity of X yetwhich also contains certain chemical structures which differ from X. Ananalogue of a naturally-occurring peptide, is a peptide which includesone or more non-naturally-occurring amino acids.

The term “mimetic refers to a compound having similar functional and/orstructural properties to another known compound or a particular fragmentof that known compound. A “mimetic” of a compound X refers to a compoundin which chemical structures of X necessary for functional activity of Xhave been replaced with other chemical structures which mimic theconformation of X. The term mimetic, and in particular, peptidomimetic,is intended to include isosteres.

The term “cyclic group”, as used herein, is intended to include cyclicsaturated or unsaturated (i.e., aromatic) group having from about 3 to10, preferably about 4 to 8, and more preferably about 5 to 7, carbonatoms. Exemplary cyclic groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and cyclooctyl. Cyclic groups may beunsubstituted or substituted at one or more ring positions. Thus, acyclic group may be substituted with halogens, alkyls, cycloalkyls,alkenyls, alkynyls, aryls, heterocycles, hydroxyls, aminos, nitros,thiols amines, imines, amides, phosphonates, phosphines, carbonyls,carboxyls, silyls, ethers, thioethers, sulfonyls, sulfonates.selenoethers, ketones, aldehydes, esters, ‘CF₃, ‘CN, or the like.

The term “heterocyclic group” is intended to include cyclic saturated orunsaturated (i.e., aromatic) group having from about 3 to 10, preferablyabout 4 to 8, and more preferably about 5 to 7, carbon atoms, whereinthe ring structure includes about one to four heteroatoms. Heterocyclicgroups include pyrrolidine, oxolane, thiolane, imidazole, oxazole,piperidine, piperazine, morpholine and pyridine. The heterocyclic ringcan be substituted at one or more positions with such substituents as,for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls,other heterocycles, hydroxyl, amino, nitro thiol, amines, imines,amides, phosphonates, phosphines, carbonyls, carboxyls, eilyls, ethers,thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, CF₃,CN, or the like. Heterocycles may also be bridged or fused to othercyclic groups as described below.

“Aryl” includes groups with aromaticity, including “conjugated,” ormulticyclic systems with at least one aromatic ring and do not containany heteroatom in the ring structure. Examples include phenyl, benzyl,1,2,3,4-tetrahydronaphthalenyl, etc.

“Heteroaryl” groups are aryl groups, as defined above, except havingfrom one to four heteroatoms in the ring structure, and may also bereferred to as “aryl heterocycles” or “heteroaromatics.” As used herein,the term “heteroaryl” is intended to include a stable 5-, 6-, or7-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclicaromatic heterocyclic ring which consists of carbon atoms and one ormore heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6heteroatoms, or e.g. 2, 3, 4, 5, or 6 heteroatoms, independentlyselected from the group consisting of nitrogen, oxygen and sulfur. Thenitrogen atom may be substituted or unsubstituted (i.e., N or NR whereinR is H or other substituents, as defined). The nitrogen and sulfurheteroatoms may optionally be oxidized (i.e., N→O and S(O)_(p), wherep=1 or 2). It is to be noted that total number of S and 0 atoms in thearomatic heterocycle is not more than 1.

Examples of heteroaryl groups include pyrrole, furan, thiophene,thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole,oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and thelike.

Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryland heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene,benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline,naphthrydine, indole, benzofuran, purine, benzofuran, deazapurine,indolizine.

In the case of multicyclic aromatic rings, only one of the rings needsto be aromatic (e.g., 2,3-dihydroindole), although all of the rings maybe aromatic (e.g., quinoline). The second ring can also be fused orbridged.

The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring can besubstituted at one or more ring positions (e.g., the ring-forming carbonor heteroatom such as N) with such substituents as described above, forexample, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl,aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety. Aryl and heteroarylgroups can also be fused or bridged with alicyclic or heterocyclicrings, which are not aromatic so as to form a multicyclic system (e.g.,tetralin, methylenedioxyphenyl).

The term “polycyclic group” as used herein is intended to refer to twoor more saturated or unsaturated (i.e., aromatic) cyclic rings in whichtwo or more carbons are common to two adjoining rings, e.g., the ringsare “fused rings”. Rings that are joined through non-adjacent atoms aretermed “bridged” rings. Each of the rings of the polycyclic group can hesubstituted with such substituents as described above, as for example,halogens, alkyls, cycloalkyls, alkenyls, alkynyls, hydroxyl, amino,nitro, thiol, amines, imines, amides, phosphonates, phosphines,carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls,selenoethers, ketones, aldehydes, esters, CF₃, CN, or the like.

As used herein, the term “modulating group” and “modifying group” areused interchangeably to describe a chemical group directly or indirectlyattached to a peptidic structure. For example, a modifying group(s) canbe directly attached by covalent coupling to the peptidic structure or amodifying group(s) can be attached indirectly by a stable non-covalentassociation.

The compounds described herein include the compounds themselves, as wellas their salts, their solvates, and their prodrugs, if applicable. Asalt, for example, can be formed between an anion and a positivelycharged group (e.g., amino) on a substituted benzene compound. Suitableanions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate,nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate,glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate,tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, andacetate (e.g., trifluoroacetate). The term “pharmaceutically acceptableanion” refers to an anion suitable for forming a pharmaceuticallyacceptable salt. Likewise, a salt can also be formed between a cationand a negatively charged group (e.g., carboxylate) on a substitutedbenzene compound. Suitable cations include sodium ion, potassium ion,magnesium ion, calcium ion, and an ammonium cation such astetramethylammonium ion. The substituted benzene compounds also includethose salts containing quaternary nitrogen atoms.

Examples of prodrugs include esters and other pharmaceuticallyacceptable derivatives, which, upon administration to a subject, arecapable of providing active substituted benzene compounds.

Additionally, the compounds of the present invention, for example, thesalts of the compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include monohydrates, dihydrates, etc. Nonlimitingexamples of solvates include ethanol solvates, acetone solvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O.

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent invention includes all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like, it being understood that not all isomers mayhave the same level of activity. In addition, a crystal polymorphism maybe present for the compounds represented by the formula. It is notedthat any crystal form, crystal form mixture, or anhydride or hydratethereof is included in the scope of the present invention. Furthermore,so-called metabolite which is produced by degradation of the presentcompound in vivo is included in the scope of the present invention.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solutions wheretautomerization is possible, a chemical equilibrium of the tautomerswill be reached. The exact ratio of the tautomers depends on severalfactors, including temperature, solvent and pH. The concept of tautomersthat are interconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g., innucleobases such as guanine, thymine and cytosine), imine-enamine andenamine-enamine. An example of ketone-enol tautomerism are as shownbelow.

A small molecule is a compound that is less than 2000 Daltons in mass.The molecular mass of the small molecule is preferably less than 1000Daltons, more preferably less than 600 Daltons, e.g., the compound isless than 500 Daltons, 400 Daltons, 300 Daltons, 200 Daltons, or 100Daltons.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

Compounds such as small molecule inhibitors, polynucleotides,polypeptides, or other agents are purified and/or isolated. Purifiedcompounds are at least 60% by weight (dry weight) the compound ofinterest. Preferably, the preparation is at least 75%, more preferablyat least 90%, and most preferably at least 99%, by weight the compoundof interest. For example, a purified compound is one that is at least90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desiredcompound by weight. Purity is measured by any appropriate standardmethod, for example, by column chromatography, thin layerchromatography, or high-performance liquid chromatography (HPLC)analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA)or deoxyribonucleic acid (DNA)) is free of the genes or sequences thatflank it in its naturally-occurring state. An “isolated” or “purified”nucleic acid molecule, polynucleotide, polypeptide, or protein, issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized. Purified also defines a degree ofsterility that is safe for administration to a human subject, e.g.,lacking infectious or toxic agents.

DETAILED DESCRIPTION

ASPH (a.k.a., AAH) is a member of the α-ketoglutarate-dependentdioxygenase family enzyme. It has a predicted molecular mass of ˜86 kDand catalyzes the hydroxylation of specific Asp (Asparate) and Asn(Asparagine) residues in EGF-like domains of certain receptor proteinssuch as Notch. Overexpression of ASPH has been observed in a broad rangeof malignant neoplasms including hepatocellular carcinoma,cholangiocellular carcinoma, pancreatic cancer, prostate cancer, breastcancer, glioblastoma, lung and colon cancer.

However, ASPH has low or negligible expression in normal adult tissuesexcept for proliferating trophoblastic cells of the placenta. In humanHCC cell lines, ASPH promotes the motility and invasiveness of tumorcells through upregulation and activation of the Notch signalingcascade. Indeed, ASPH overexpression is reported to be a poor prognosticfactor for patients with HCC and predicts early disease recurrence andreduced survival. Especially in colon cancer, there is a significantassociation between poor surgical outcome and ASPH expression, which isconsidered an independent risk factor indicating poor prognosis withthis disease. Another tumor with high ASPH expression is pancreaticcancer (PC) which is the fourth leading cause of cancer mortality in theUnited States with a five-year survival rate of 5-6%. PC is an extremelyaggressive tumor refractory to most therapies. There is a need to definethe molecular pathogenesis of PC and develop more effective treatmentstrategies. Signaling pathways mediated by ASPH participate in thegrowth and metastasis of PC during oncogenesis. This surprisingdiscovery on the role of ASPH overexpression in PC pathogenesisindicates that ASPH is a molecular target for therapy and thatinhibition of ASPH in this type of cancer leads to clinical benefit.

Accordingly, in one aspect, the invention features an asparatyl(asparaginyl) beta-hydroxylase (ASPH) inhibitory compound for use in amethod of reducing proliferation, migration, invasion, or metastasis ofa tumor cell in the treatment of cell proliferative disorder, comprisingcontacting said tumor cell with the ASPH inhibitory compound, whereinthe ASPH inhibitory compound is of Formula Ia or Ib:

or a salt, ester, metabolite, prodrug, or solvate thereof, wherein

Ar¹ is substituted or unsubstituted C₆-C₂₀ aryl or 5 to 20-memberedheteroaryl;

X is C(O), C(S), or S(O)₂;

W¹ is a single bond, O, CR⁵⁰R⁵¹, or NR⁵² when X is CO and W¹ is a singlebond, CR⁵⁰R⁵¹, or NR⁵² when X is SO₂; and

each of R⁵⁰, R⁵¹, R⁵², and R⁵³ independently is selected from the groupconsisting of hydrogen, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted C₂-C₆ alkenyl, substituted or unsubstitutedC₂-C₆ alkynyl, substituted or unsubstituted C₆-C₂₀ aryl, substituted orunsubstituted C₇-C₂₆ arylalkyl, substituted or unsubstituted 5 to20-membered heteroaryl, and substituted or unsubstituted 6-26 memberedheteroarylalkyl.

In one embodiment, the compound for said use is of Formula Ia, or asalt, ester, metabolite, prodrug, or solvate thereof. The compound ofFormula Ia may have one or more of the following features whenapplicable.

For example, the compound is of Formula IIa:

or a salt, ester, metabolite, prodrug, or solvate thereof, wherein

each of Ar¹ and Ar² independently is unsubstituted C₆-C₁₄ aryl,unsubstituted 5 to 14-membered heteroaryl, or C₆-C₁₄ aryl or 5 to14-membered heteroaryl each substituted with one or more substituentsselected from the group consisting of halo, CN, NO₂, NO, N₃, OR_(a),NR_(a)R_(b), C(O)R_(a), C(O)OR_(a), C(O)NR_(a)R_(b), NR_(b)C(O)R_(a),—S(O)_(b)R_(a), —S(O)_(b)NR_(a)R_(b), or R_(S1), in which R_(S1) isC₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 5- or 6-membered heteroaryl, or 4 to 12-membered heterocycloalkyl,b is 0, 1, or 2, each of R_(a) and R_(b), independently is H or R_(S2),and R_(S2) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or6-membered heteroaryl; and each of R_(S1) and R_(S2), is optionallysubstituted with one or more substituents selected from the groupconsisting of halo, OH, oxo, C(O)OH, C(O)O—C₁-C₆ alkyl, CN, C₁-C₆ alkyl,C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, and 5- or6-membered heteroaryl.

For example, R⁵³ is unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substitutedwith one or more substituents selected from halo, OH, CN, and amino.

For example, X is S(O)₂ and W¹ is CR⁵⁰R⁵¹.

For example, X is S(O)₂ and W¹ is a single bond.

For example, X is C(O) and W¹ is O, or X is C(S) and W¹ is NR⁵².

For example, each of R⁵⁰, R⁵¹, and R⁵² independently is H, unsubstitutedC₁-C₆ alkyl, or C₁-C₆ alkyl substituted with one or more substituentsselected from halo, OH, CN, and amino.

For example, each of Ar¹ and Ar² independently is phenyl, naphthyl, or 5to 10-membered heteroaryl, each of which is optionally substituted withone or more substituents selected from the group consisting of halo, CN,NO₂, NO, N₃, OR_(a), NR_(a)R_(b), C(O)R_(a), C(O)OR_(a),C(O)NR_(a)R_(b), NR_(b)C(O)R_(a), —S(O)_(b)R_(a), —S(O)_(b)NR_(a)R_(b),or R_(S1), in which R_(S1) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5- or 6-membered heteroaryl, or 4 to12-membered heterocycloalkyl, b is 0, 1, or 2, each of R_(a) and R_(b),independently is H or R_(S2), and R_(S2) is C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, or 5- or 6-membered heteroaryl; and each of R_(S1) andR_(S2), is optionally substituted with one or more substituents selectedfrom the group consisting of halo, OH, oxo, C(O)OH, C(O)O—C₁-C₆ alkyl,CN, C₁-C₆ alkyl, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl.

For example, each of Ar¹ and Ar² independently is phenyl, naphthyl, or 5to 10-membered heteroaryl, each of which is optionally substituted withone or more substituents selected from the group consisting of halo, CN,NO₂, NO, N₃, OR_(a), NR_(a)R_(b), C(O)R_(a), C(O)OR_(a), or R_(S1), inwhich R_(S1) is C₁-C₆ alkyl, each of R_(a) and R_(b), independently is Hor R_(S2), and R_(S2) is C₁-C₆ alkyl; and each of R_(S1) and R_(S2), isoptionally substituted with one or more substituents selected from thegroup consisting of halo, OH, C₁-C₆ alkoxyl, amino, mono-C₁-C₆alkylamino, and di-C₁-C₆ alkylamino.

For example, each of Ar¹ and Ar² independently is selected from phenyl,1-naphthyl, 2-naphthyl, 2-furanyl, 2-thiazolyl, 2-pyridyl, 3-pyridyl,4-pyridyl, 2-quinolinyl, 3-quinolinyl, 4-quinolinyl, 2-chlorophenyl,3-chlorophenyl, 4-chlorophenyl, 2-fluorophenyl, 3-fluorophenyl,4-fluorophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl,4-trifluoromethylphenyl, 2-cyanophenyl, 3-cyanophenyl, 4-cyanophenyl,3-carboxymethylphenyl, 2-methoxyphenyl, 3-methoxyphenyl,4-methoxyphenyl, 2,3-dichlorophenyl, 2,4-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl,3,4-difluorophenyl, 3,5-difluorophenyl, 2,3-dimethoxyphenyl,2,4-dimethoxyphenyl, 2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl,3,5-dimethoxyphenyl, 2-chloro-6-fluorophenyl, 3-chloro-4-fluorophenyl,2-chloro-4-fluorophenyl, 4-chloro-3-fluorophenyl,3-chloro-2-fluorophenyl, 2-chloro-5-fluorophenyl,4-chloro-2-fluorophenyl, and 5-chloro-2-fluorophenyl.

In another embodiment, the compound for said use is of Formula Ib, or asalt, ester, metabolite, prodrug, or solvate thereof. The compound ofFormula Ib may have one or more of the following features whenapplicable.

For example, R⁵³ is unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substitutedwith one or more substituents selected from halo, OH, CN, and amino.

For example, R⁵³ is unsubstituted methyl or ethyl.

For example, X is S(O)₂ and W¹ is CR⁵⁰R⁵¹.

For example, X is S(O)₂ and W¹ is a single bond.

For example, X is C(O) and W¹ is O, or X is C(S) and W¹ is NR⁵².

For example, each of R⁵⁰, R⁵¹, and R⁵² independently is H, unsubstitutedC₁-C₆ alkyl, or C₁-C₆ alkyl substituted with one or more substituentsselected from halo, OH, CN, and amino.

For example, Ar¹ is phenyl, naphthyl, or 5 to 10-membered heteroaryl,each of which is optionally substituted with one or more substituentsselected from the group consisting of halo, CN, NO₂, NO, N₃, OR_(a),NR_(a)R_(b), C(O)R_(a), C(O)OR_(a), C(O)NR_(a)R_(b), NR_(b)C(O)R_(a),—S(O)_(b)R_(a), —S(O)_(b)NR_(a)R_(b), or R_(S1), in which R_(S1) isC₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 5- or 6-membered heteroaryl, or 4 to 12-membered heterocycloalkyl,b is 0, 1, or 2, each of R_(a) and R_(b), independently is H or R_(S2),and R_(S2) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or6-membered heteroaryl; and each of R_(S1) and R_(S2), is optionallysubstituted with one or more substituents selected from the groupconsisting of halo, OH, oxo, C(O)OH, C(O)O—C₁-C₆ alkyl, CN, C₁-C₆ alkyl,C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, and 5- or6-membered heteroaryl.

For example, Ar¹ is phenyl, naphthyl, or 5 to 10-membered heteroaryl,each of which is optionally substituted with one or more substituentsselected from the group consisting of halo, CN, NO₂, NO, N₃, OR_(a),NR_(a)R_(b), C(O)R_(a), C(O)OR_(a), or R_(S1), in which R_(S1) is C₁-C₆alkyl, each of R_(a) and R_(b), independently is H or R_(S2), and R_(S2)is C₁-C₆ alkyl; and each of R_(S1) and R_(S2), is optionally substitutedwith one or more substituents selected from the group consisting ofhalo, OH, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, and di-C₁-C₆alkylamino.

For example, Ar¹ is selected from phenyl, 1-naphthyl, 2-naphthyl,2-furanyl, 2-thiazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-quinolinyl,3-quinolinyl, 4-quinolinyl, 2-chlorophenyl, 3-chlorophenyl,4-chlorophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl,2-trifluoromethylphenyl, 3-trifluoromethylphenyl,4-trifluoromethylphenyl, 2-cyanophenyl, 3-cyanophenyl, 4-cyanophenyl,3-carboxymethylphenyl, 2-methoxyphenyl, 3-methoxyphenyl,4-methoxyphenyl, 2,3-dichlorophenyl, 2,4-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl,3,4-difluorophenyl, 3,5-difluorophenyl, 2,3-dimethoxyphenyl,2,4-dimethoxyphenyl, 2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl,3,5-dimethoxyphenyl, 2-chloro-6-fluorophenyl, 3-chloro-4-fluorophenyl,2-chloro-4-fluorophenyl, 4-chloro-3-fluorophenyl,3-chloro-2-fluorophenyl, 2-chloro-5-fluorophenyl,4-chloro-2-fluorophenyl, and 5-chloro-2-fluorophenyl.

In one embodiment, said tumor cell expresses ASPH.

In certain embodiments, said cell proliferative disorder comprisesPancreatic Cancer, Hepatocellular Cancer, Cholangiocarcinoma, Lungcancer, Colon Cancer, Breast Cancer, Prostatic Cancer, and Glioblastoma.

In one embodiment, said compound is administered intravenously, orally,or subcutaneously.

In one embodiment, said compound is administered at a dose of 0.01 to 50milligrams/kilogram of body weight.

ASPH is a highly conserved cell-surface protein in hepatocellularcarcinoma (HCC). Both the liver and the pancreas are derived from anearly progenitor cell type and ASPH is expressed in embryo but not inadult tissues. ASPH re-expression was observed in human PC tissuemicroarrays by immunohistochemical staining (IHS) as shown in FIGS.1A-E. High level cell surface localization of ASPH was present in 101 of104 (97%) pancreatic ductal adenocarcinoma with negligible expression innormal pancreas, and other adult human tissues. ASPH enhances cellmigration, invasion, and metastasis in HCC and also PC. Activation ofNotch signaling by ASPH is a final effector mechanism responsible forgeneration of this highly aggressive and malignant phenotype.

Biological Properties of ASPH as a Cellular Target

The regulation, expression, and function of ASPH has been observed inmany tumors (U.S. Pat. Nos. 6,835,370; 7,094,556; 6,812,206; 6,815,415;6,797,696; 6,783,758; and U.S. Published Patent Application No.2005-0123545; hereby incorporated by reference) and ASPH has been foundto be overexpressed in pancreatic ductal adenocarcinoma (PC) indicatingthat it is a therapeutic target for treatment of PC. ASPH catalyzespost-translational hydroxylation of β-carbons of specific aspartate andasparaginyl residues in epidermal growth factor (EGF)-like domainsresiding in proteins such as Notch and Jagged (JAG) which are involvedin cell growth, differentiation, cellular migration, adhesion, andmotility. The catalytic activity resides in the C-terminus and isconferred by the ⁶⁷⁵His residue; mutation to an alanine abolishes ASPHenzymatic and transforming activity. ASPH is overexpressed in tumorsderived from the endoderm such as liver, pancreas, colon and lung, andtranslocates from the endoplasmic reticulum (ER) to the plasma membranewhere it becomes accessible to the extracellular environment. It hasnegligible to very low expression in normal human tissue with thenotable exception of the placenta which is an invasive tissue, and itsexpression there is robust.

Compounds are administered directly into a tumor site or systemically toinhibit ASPH hydroxylase activity.

cDNA sequence of human ASPH (SEQ ID NO: 3)cggaccgtgc aatggcccag cgtaagaatg ccaagagcag cggcaacagc agcagcagcg 61gctccggcag cggtagcacg agtgcgggca gcagcagccc cggggcccgg agagagacaa 121agcatggagg acacaagaat gggaggaaag gcggactctc gggaacttca ttcttcacgt 181ggtttatggt gattgcattg ctgggcgtct ggacatctgt agctgtcgtt tggtttgatc 241ttgttgacta tgaggaagtt ctaggaaaac taggaatcta tgatgctgat ggtgatggag 301attttgatgt ggatgatgcc aaagttttat taggacttaa agagagatct acttcagagc 361cagcagtccc gccagaagag gctgagccacacactgagcc cgaggagcag gttcctgtgg 421aggcagaacc ccagaatatc gaagatgaag caaaagaaca aattcagtcc cttctccatg 481aaatggtaca cgcagaacat gttgagggag aagacttgca acaagaagat ggacccacag 541gagaaccaca acaagaggat gatgagtttc ttatggcgac tgatgtagat gatagatttg 601agaccctgga acctgaagta tctcatgaag aaaccgagca tagttaccac gtggaagaga 661cagtttcaca agactgtaat caggatatgg aagagatgat gtctgagcag gaaaatccag 721attccagtga accagtagta gaagatgaaa gattgcacca tgatacagat gatgtaacat 781accaagtcta tgaggaacaa gcagtatatg aacctctaga aaatgaaggg atagaaatca 841cagaagtaac tgctccccct gaggataatc ctgtagaaga ttcacaggta attgtagaag 901aagtaagcat ttttcctgtg gaagaacagc aggaagtacc accagaaaca aatagaaaaa 961cagatgatcc agaacaaaaa gcaaaagtta agaaaaagaa gcctaaactt ttaaataaat 1021ttgataagac tattaaagct gaacttgatg ctgcagaaaa actccgtaaa aggggaaaaa 1081ttgaggaagc agtgaatgca tttaaagaac tagtacgcaa ataccctcag agtccacgag 1141caagatatgg gaaggcgcag tgtgaggatg atttggctga gaagaggaga agtaatgagg 1201tgctacgtgg agccatcgag acctaccaag aggtggccag cctacctgat gtccctgcag 1261acctgctgaa gctgagtttg aagcgtcgct cagacaggca acaatttcta ggtcatatga 1321gaggttccct gcttaccctg cagagattag ttcaactatt tcccaatgat acttccttaa 1381aaaatgacct tggcgtggga tacctcttga taggagataa tgacaatgca aagaaagttt 1441atgaagaggt gctgagtgtg acacctaatg atggctttgc taaagtccat tatggcttca 1501tcctgaaggc acagaacaaa attgctgaga gcatcccata tttaaaggaa ggaatagaat 1561ccggagatcc tggcactgat gatgggagat tttatttcca cctgggggat gccatgcaga 1621gggttgggaa caaagaggca tataagtggt atgagcttgg gcacaagaga ggacactttg 1681catctgtctg gcaacgctca ctctacaatg tgaatggact gaaagcacag ccttggtgga 1741ccccaaaaga aacgggctac acagagttag taaagtcttt agaaagaaac tggaagttaa 1801tccgagatga aggccttgca gtgatggata aagccaaagg tctcttcctg cctgaggatg 1861aaaacctgag ggaaaaaggg gactggagcc agttcacgct gtggcagcaa ggaagaagaa 1921atgaaaatgc ctgcaaagga gctcctaaaa cctgtacctt actagaaaag ttccccgaga 1981caacaggatg cagaagagga cagatcaaat attccatcat gcaccccggg actcacgtgt 2041ggccgcacac agggcccaca aactgcaggc tccgaatgca cctgggcttg gtgattccca 2101aggaaggctg caagattcga tgtgccaacg agaccaggac ctgggaggaa ggcaaggtgc 2161tcatctttga tgactccttt gagcacgagg tatggcagga tgcctcatct ttccggctga 2221tattcatcgt ggatgtgtgg catccggaac tgacaccaca gcagagacgc agccttccag 2281caatttagca tgaattcatg caagcttggg aaactctgga gaga(SEQ ID NO:3 ; GenBank Accession No. 583325; codon encoding initiatingmethionine is underlined). Amino acid sequence of human ASPH(SEQ ID NO: 4)MAQRKNAKSS GNSSSSGSGS GSTSAGSSSP GARRETKHGG HKNGRKGGLS GTSFFTWFMV 61IALLGVWTSV AVVWFDLVDY EEVLGKLGIY DADGDGDFDV DDAKVLLGLK ERSTSEPAVP 121PEEAEPHTEP EEQVPVEAEP QNIEDEAKEQ IQSLLHEMVH AEHVEGEDLQ QEDGPTGEPQ 181QEDDEFLMAT DVDDRFETLE PEVSHEETEH SYHVEETVSQ DCNQDMEEMM SEQENPDSSE 241PVVEDERLHH DTDDVTYQVY EEQAVYEPLE NEGIEITEVT APPEDNPVED SQVIVEEVSI 301FPVEEQQEVP PETNRKTDDP EQKAKVKKKK PKLLNKFDKT IKAELDAAEK LRKRGKIEEA 361VNAFKELVRK YPQSPRARYG KAQCEDDLAE KRRSNEVLRG AIETYQEVAS LPDVPADLLK 421LSLKRRSDRQ QFLGHMRGSL LTLQRLVQLF PNDTSLKNDL GVGYLLIGDN DNAKKVYEEV 481LSVTPNDGFA KVHYGFILKA QNKIAESIPY LKEGIESGDP GTDDGRFYFH LGDAMQRVGN 541KEAYKWYELG HKRGHFASVW QRSLYNVNGL KAQPWWTPKE TGYTELVKSL ERNWKLIRDE 601GLAVMDKAKG LFLPEDENLR EKGDWSQFTL WQQGRRNENA CKGAPKTCTL LEKFPETTGC 661RRGQIKYSIM HPGTHVWPHT GPTNCRLRMH LGLVIPKEGC KIRCANETRT WEEGKVLIFD 721DSFEHEVWQD ASSFRLIFIV DVWHPELTPQ QRRSLPAI(SEQ ID NO:4; GenBank Accession No. S83325; His motif is underlined; conservedsequences within the catalytic domain are designated by bold type)

Methods of inhibiting tumor growth also include administering a compoundwhich inhibits HAAH hydroxylation of a NOTCH polypeptide. For example,the compound inhibits hydroxylation of an EGF-like cysteine-rich repeatsequence in a NOTCH polypeptide, e.g., one containing the consensussequence

(SEQ ID NO: 2) CDXXXCXXKXGNGXCDXXCNNAACXXDGXDC.

Polypeptides containing an EGF-like cysteine-rich repeat sequence areadministered to block hydroxylation of endogenous NOTCH.

ASPH is expressed in many organs during embryogenesis presumably topromote cell motility and migration for cell patterning and organdevelopment; its expression is “shut off” in the adult only to re-emergeduring oncogenesis where its function may be required for generation ofmalignant phenotypes. It appears not to be overexpressed during cellproliferation; however, there is low-level expression in dysplasticductal cells of pancreatic intraepithelial lesions (PanINs) as well asdysplastic hepatocytes in hepatitis B (HBV) and C (HCV) infected liver.Transcriptional regulation of ASPH is provided by tripartite signalingpathways IN/IGF1/IRS1/MAPK/ERK, IN/IGF1/IRS1/PI3K/AKT, andWNT/β-Catenin. Post-transcriptional regulation of ASPH is mediated byphosphorylation of GSK-3β-related motifs located in the N-terminalregion of the molecule. One mechanism by which ASPH exerts its effectorfunction is by activating downstream Notch signaling to promote cellmigration and invasion.

ASPH Expression in Human Tumors

Table 1 details the overexpression of ASPH at the protein and RNA levelas determined by IHS and qRT-PCR respectively in various human tumorsindicating that it is a therapeutic target for a variety of human solidmalignancies with a poor prognosis. FIGS. 1 and 2 show examples of ASPHprotein expression by IHS.

TABLE 1 Expression of ASPH in Human Tumors Compared to Normal Tissue byIHS and qRT-PCR Number Tumor type Number Positive (%) Pancreatic Cancer101 98  (97%) Hepatocellular Cancer 95 87  (92%) Cholangiocarcinoma 2020 (100%) Lung 16 16 (100%) Colon Cancer 10 6  (60%) Breast Cancer 17 17(100%) Prostatic Cancer 32 30  (94%) Glioblastoma 5 5 (100%)

Oncogenic Role of ASPH β-Hydroxylase Activity

The C-terminus of ASPH contains amino acid (AA) sequence of thecatalytic site (M⁶⁷⁰HPGFH⁶⁷⁵) and its sequence is identical in human,rat, mouse, and cattle. The H⁶⁷⁵AA is specifically involved in Fe²⁺coordination and critical for its enzymatic activity, also highlyconserved in the chicken and fly. A H⁶⁷⁵R mutation reduces β-hydroxylaseactivity to <1% of wild-type protein while H⁶⁷⁵D reduces it to 20%. Inthis context the H⁶⁷⁵R mutant protein loses the ability to promote cellproliferation, motility, migration, invasion, colony formation in softagar, as well as metastasis and tumor formation in nude mice compared tothe “wild-type” sequence and it also can function as a dominant negativemutant to inhibit the function of the endogenous “Wild-Type” protein.These findings indicate that inhibition of β-hydroxylase activitypromotes anti-tumor effects. The crystal structure of the catalytic siteregion has been elucidated and is available in the public database (RCSBprotein database; code 3RCQ). Small molecule inhibitors for use asanti-tumor agents were identified by their ability to fit into thecatalytic site region of ASPH and inhibit ASPH enzymatic activity.

Generation of a Small Molecule Inhibitor (SMI) of ASPH EnzymaticActivity

Compounds of the present invention can be prepared in a variety of waysusing commercially available starting materials, compounds known in theliterature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5^(th) edition, John Wiley & Sons: New York, 2001;Greene, T.W., Wuts, P.G. M., Protective Groups in Organic Synthesis,3^(rd) edition, John Wiley & Sons: New York, 1999; R. Larock,Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieserand M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, JohnWiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagentsfor Organic Synthesis, John Wiley and Sons (1995), incorporated byreference herein, are useful and recognized reference textbooks oforganic synthesis known to those in the art. The following descriptionsof synthetic methods are designed to illustrate, but not to limit,general procedures for the preparation of compounds of the presentinvention.

In the reaction schemes described herein, multiple stereoisomers may beproduced. When no particular stereoisomer is indicated, it is understoodto mean all possible stereoisomers that could be produced from thereaction. A person of ordinary skill in the art will recognize that thereactions can be optimized to give one isomer preferentially, or newschemes may be devised to produce a single isomer. If mixtures areproduced, techniques such as preparative thin layer chromatography,preparative HPLC, preparative chiral HPLC, or preparative SFC may beused to separate the isomers.

Scheme 1 above shows the synthetic strategies for compounds of FormulaIa. Reactions a-f are as follows: (a) KCN, glyoxal, Na₂CO₃, H₂O; (b)ClSO₂R, TEA, THF; (c) ClSO₂CH₂Ph, TEA, THF; (d) ClSO₂CH₂Ar², TEA, THF;(e) ClCO₂Ar², TEA, THF; (f) Ar²NCS, Na₂CO₃, H₂O.

Synthesis and Characterization of Novel SMI for ASPH.

Based on the crystal structure of the ASPH catalytic site, computergenerated drug design was performed that has led to the synthesis of aseries of parent compounds and derivatives to fit into the pocket of thecatalytic site and inhibit the β-hydroxylase activity. Parent compounds(Compound 307 {7c or MO-I-1000}, and Compound 310 {8c or MO-I-1100})were synthesized and examined for inhibition of β-hydroxylase activityusing a high throughput screening assay. Synthesis of ASPH inhibitorswas accomplished in two steps. The first step was a three componentreaction including an aromatic aldehyde, glyoxal bisulfate additionproduct, and potassium cyanide to yield an arylhydroxytetronimide.

In the second step the arylhydroxytetronimide was sulfonylated withphenylmethanesulfonyl chloride in dry tetrahydrofuran to yield compoundsof Formula Ia (Scheme 1). Compounds were characterized by 1H and 13Cnuclear magnetic resonance, high resolution mass spectroscopy, highperformance liquid chromatography, infra-red spectroscopy, meltingpoint, elemental analysis and binding to ASPH by isothermal titrationcalorimetry.

Existing Structure Activity Relationship (SAR) Exploration.

Compound 403 {3c or MO-I-1000}, Compound 310 {8c or MO-I-1100}, Compound404 {4c or MO-I-400} and Compound 405 {5c or MO-I-500} were identifiedas hits. This led to identification of a mixture of enantiomers as thelead compounds 2 and 3 and the additional recognition that the Compound405 {5c or MO-I-500} compound demonstrated biologic activity through itsaction on ASPH enzymatic activity.

Characterization of a High throughput Enzymatic Assay for ASPH Activity.

EGF and EGF-like domains are well known in the art and generally includesix cysteine residues which have been shown (in EGF) to be involved indisulfide bonds. The main structure is a two-stranded beta-sheetfollowed by a loop to a C-terminal short two-stranded sheet. Subdomainsbetween the conserved cysteines vary in length. Examples include thosedescribed in Davis, C G, 1990, New Biol. 2(5):410-9; Blomquist et al.,1984, Proc Natl Acad Sci USA. 81(23):7363-7; Hommel et al., 1002, J MolBiol. 227(1):271-82; Doolittle et al., 1984, Nature. 307(5951):558-60;Appella et al., 1988, FEBS Lett. 231(1):1-4; Sorkin A., 2001, BiochemSoc Trans. Aug;29(Pt 4):480-4; each of which is hereby incorporated byreference).

FIGS. 3A-C represents the strategy for development and characterizationof the performance of an assay to measure ASPH enzymatic activity. FIG.3A describes the biochemical reaction catalyzed by ASPH. FIG. 3B depictsthe read-out of the assay. Protein lysates were extracted from FOCUScells (high level of ASPH cell surface expression) treated with 1-10 μMof each parent compound for 24 hours. The lysates were added into 96well-plates coated with ASPH monoclonal antibodies (mAbs) to add anantigen specific (ASPH) capture step to the assay design. Afterincubation and washing, only ASPH is captured on each well. The reactionis carried out with 60 μM of an EGF domain containing 39AA peptide, 100μM FeCl₂ and 40 μM ¹⁴C labeled α-ketoglutarate were added into eachwell. The ¹⁴CO₂ was captured on a glass fiber membrane soaked inCa(OH)₂. Radioactivity captured was quantified by a phosphor-imager asshown in FIG. 3C. FIG. 3D shows that Compound 310 {8c or MO-I-1100} at aconcentration of 1 μM substantially inhibits β-hydroxylase activity. Thecompound was further characterized for clinical use as an anti-tumoragent for PC and HCC and other ASPH-expressing tumors.

PC Growth Inhibition in a Preclinical Murine Model

After demonstrating that Compound 310 {8c or MO-I-1100} inhibitedβ-hydroxylase activity using the ASPH specific mAb capture enzymaticassay, its activity as an anti-tumor agent in a nude mouse model ofsubcutaneous (s.c.) tumor growth was evaluated. The HPAFII AsPC-1 humanPC cell line which expresses a high level of ASPH was implanted (5×10⁶cells s.c.) into the back of nude mice. Tumors were allowed to grow toapproximately 100 mm³ after one week, and Compound 310 {8c or MO-I-1100}was then administered intraperitoneal (i.p.) at a concentration of 50mg/kg. The treatment regimen as shown in FIG. 4A-B included i.p.injections on a daily basis for five days followed by every other dayuntil the experiment was terminated due to the large size of tumorsobserved in the control group that received a DMSO vehicle injection.FIG. 4A-B demonstrates the growth rate and substantial inhibition ofHPAFII tumor formation over the course of the study. There were 15 nudemice in each group (control vs. treatment). Therefore, this studydemonstrates that the compound (Compound 310 {8c or MO-I-1100}) wasactive in vitro inhibiting ASPH β-hydroxylase activity, and performedwell in vivo as an anti-tumor agent.

In Vitro Effects of ASPH Inhibitors for Cell Proliferation andMetabolism:

Studies with Compound 310 {8c or MO-I-1100}

A MTT assay was performed to evaluate the effect of Compound 310 {8c orMO-I-1100} on cell proliferation and viability. MTT is reduced to purpleformazan in living cells. FOCUS cells were used as a high ASPHexpressing human HCC cell line. The results shows that treatments withCompound 310 {8c or MO-I-1100} for 24 hours dose-dependently decreasedthe OD (optical density) value in FOCUS cells (FIGS. 5A-B). However, inNIH-3T3 cells, which is a mouse embryo fibroblast cell line notexpressing ASPH, Compound 310 {8c or MO-I-1100} had no effect on the ODvalue of MTT indicating that Compound 310 {8c or MO-I-1100} is highlyspecific for the β-hydroxylase activity of ASPH and did not affect cellsthat lacked ASPH expression. The MTT assay measures cellular metabolicactivity via NAD(P)H-dependent cellular oxidoreductase enzymes. However,as shown in FIG. 5C, Compound 310 {8c or MO-I-1100} also decreased cellviability in human HCC cell lines at 5 μM (FOCUS, Hep3B, HepG2 and Huh7)which express ASPH (inhibition rate 37.9%, 60%, 59% and 50%,respectively) but not NIH 3T3 cells with no expression of ASPH. In orderto evaluate the effect of long-term exposure of Compound 310 {8c orMO-I-1100}, the colony formation assay (an assay of malignant potentialand phenotype) in which cells were treated with inhibitor for 3 weekswas performed. Treatment with Compound 310 {8c or MO-I-1100} resulted inreduced colony formation at 5 tM concentration (inhibition rate 36.8%)(FIGS. 6A-B).

Studies on Compound 405 {5c or MO-I-500}

We performed similar studies using the Compound 405 {5c or MO-I-500}compound. Similar to the findings in Compound 310 {8c or MO-I-1100}, weobserved that Compound 405 {5c or MO-I-500} at 5 μM also inhibited ASPHenzymatic activity as measured by the ¹⁴CO₂-ketoglutarate-dependentcapture assay. The structure of Compound 405 {5c or MO-I-500} is quitedifferent than Compound 310 {8c or MO-I-1100} and the results of thebiological activity of Compound 405 {5c or MO-I-500} is shown in FIGS.7A-B. Additional experiments measured the in vitro effects of Compound405 {5c or MO-I-500} on cell proliferation and metabolism as shown inFIGS. 8A-D. In contrast to Compound 310 {8c or MO-I-1100} effects,Compound 405 {5c or MO-I-500} has striking effects on cell proliferationand metabolic activity at micromolar concentrations in both FOCUS cells(which contain high levels of ASPH cell surface expression) and NIH-3T3cells which do not. These findings indicate that Compound 405 {5c orMO-I-500} has hydroxylase inhibitory activity but also probably inhibitsother hydroxylases or proteins that may be important for cell viabilityand growth as well since NIH 3T3 cells lacking ASPH were susceptible toits inhibitory effects. More striking was the colony formation assaythat showed that 1.25 μM concentrations of Compound 405 {5c or MO-I-500}had substantial inhibitory effects on colony formation of FOCUS HCCcells indicating high potency in this assay of cellular transformation.In summary both Compound 310 {8c or MO-I-1100} and Compound 405 {5c orMO-I-500} inhibit ASPH β-hydroxylase activity, cell viability andmetabolism, as well as colony formation in soft agar but Compound 310{8c or MO-I-1100} is more highly specific for the β-hydroxylase of ASPHas shown in FIGS. 5A-C.

In Vitro Effects of ASPH Inhibitor for Anchorage-Independent CellGrowth: Studies on Compound 310 {8c or MO-I-1100}

The effect of Compound 310 {8c or MO-I-1100} on anchorage independentgrowth in soft agar was evaluated. The ability of forming colonospherein soft agar is considered to be a rigid test for tumorigenic potential.ASPH expression results in cells acquiring the ability to formcolonospheres in soft agar. These results indicated that ASPH plays akey role in establishing tumor, growth, invasion and metastasis in vivo.In order to evaluate the effect of ASPH inhibitor for anchorageindependent colony formation, FOCUS cells were incubated in soft agarwith or without this ASPH enzymatic inhibitor. After 3 weeks incubation,Compound 310 {8c or MO-I-1100} reduced colonosphere formation of FOCUScells (FIG. 9A). As shown in FIG. 9B,C treatment with Compound 310 {8cor MO-I-1100} produced a dose-dependent and highly significant reductionboth in number and size of the colonies after 3 weeks of culture. Thisassay is a standard method to monitor malignant growth that reflects theability to form tumors that grow aggressively in vivo. These resultsconfirm that Compound 310 {8c or MO-I-1100} impairs the generation of amalignant phenotype in vitro.

Studies with Compound 405 {5c or MO-I-500}

The effect of Compound 405 {5c or MO-I-500} on anchorage independentcell growth was evaluated as shown in FIGS. 10A-B. It was striking that1 μM exposure of HCC cells grown in soft agar dramatically reduced thenumber of colonies formed, and the colony size in a dose-dependentmanner. Thus, Compound 405 {5c or MO-I-500 } had very similar but morepotent effects on anchorage-independent cell growth as did Compound 310{8c or MO-I-1100} for this assay of cellular transformation thatcorrelates well with tumor formation in vivo.

In Vitro Effects of ASPH Inhibitors for Cell Motility and Invasivenessin Human HCC Cell:

Studies with Compound 310 {8c or MO-I-1100}

The β-hydroxylase activity is required for ASPH to mediate its effectson cell motility. Directional motility was measured using Boydenchamber-type culture inserts equipped with porous membranes. FOCUS cellswere pretreated with 5 μM of Compound 310 {8c or MO-I-1100} and Compound405 {Sc or MO-I-500} β-hydroxylase inhibitor for 24 hours, and thenplaced into the upper chamber. Migration was allowed to proceed for 30min. ATPLite was used to quantify viable cell density. The cells in theupper well and upper surface of membrane reflects the number ofnon-migrating cells, luminescence measured at the bottom surface of themembrane reflects the number of migrating and non-adherent cells andluminescence measured in the bottom well was reflect migrating andnon-adherent cells. As shown in FIG. 11A-C total migrated cells werereduced following Compound 310 {8c or MO-I-1100} treatment but thepopulation of non-adherent cells was unchanged; the migrating andadherent cells were significantly reduced. Cell invasiveness wasassessed by invasion assay using matrigel coated membrane, in whichcells were allowed to proceed for 6 hours; those found adhered on thebottom surface of membrane were regarded as invading cells. Compound 310{8c or MO-I-1100} treatment FOCUS cells significantly reducedinvasiveness compared to cells incubated with DMSO as a control (FIG.11D). Results demonstrated that Compound 310 {8c or MO-I-1100} inhibitedcell motility, migration and invasiveness of human HCC cells.

Studies with Compound 405 {5c or MO-I-500}

In vivo effects of ASPH inhibitors on cell motility and invasivenesswere evaluated using the human HCC FOCUS cell line. As shown in FIGS.12A-B, Compound 405 {5c or MO-I-500} had a pronounced effect on cellmotility and invasion as well. Note that Compound 405 {5c or MO-I-500}was slightly more potent inhibiting cell motility and invasion thanCompound 310 {8c or MO-I-1100}, but both were highly active in these twoassays that characterize the malignant phenotype. Thus, these smallmolecule inhibitors of ASPH-β-hydroxylase have a profound effect on thefunction of the metastatic phenotype by reducing the ability of tumorcells to migrate and invade, and thus substantially alter their biologicfunction and metastatic potential.

In Vivo Effects of an ASPH Inhibitor, Compound 310 {8c or MO-I-1100}, onSubcutaneous Xenograft Development and Growth of Human HepatocellularCarcinoma.

The human HCC cell line FOCUS is known to be a highly aggressive tumorforming cell line in vivo. To investigate in vivo anti-tumor efficacy ofthe ASPH inhibitor, Compound 310 {8c or MO-I-1100} at 20 mg/kg per daywas administered on 5 consecutive days for 2 weeks and every other daythereafter. As shown in FIGS. 13A-B, the administration of Compound 310{8c or MO-I-1100} significantly reduced HCC subcutaneous xenograftgrowth. The mean tumor volumes were substantially decreased by treatmentwith Compound 310 {8c or MO-I-11001} on day 12 following treatment, andtumor volumes in treated mice were reduced an average of 31.7% comparedto those observed in control mice (FIG. 15B). None of the Compound 310{8c or MO-I-1100} treated mice showed signs of wasting or other adverseeffects relative to control mice. Thus, Compound 310 {8c or MO-I-1100}was tolerated well at this dose level where striking antitumor efficacywas observed. Thus, a specific β-hydroxylase inhibitor such as Compound310 {8c or MO-I-1100} substantially reduced tumor growth of HCC as shownin FIGS. 15A-B but also inhibited the development and growth ofpancreatic cancer as well (FIG. 6A-B) since the tumor also has highlevel expression of ASPH. Such findings indicate that any ASPHexpressing human tumor are responsive to these specific β-hydroxylaseinhibitors. These ASPH inhibitor compounds represent a class ofanti-tumor agents that has substantial anti-tumor activity against alarge number of solid human tumors known to have a dismal prognosis.

ASPH is overexpressed on the cell surface of human tumor cells withinsolid tumors and has low or negligible expression in normal humantissues. ASPH expression is present in most, if not all, tumor cells.ASPH is also exposed to the extracellular environment which makes it anexcellent therapeutic target since it has easy access to small moleculeinhibitors of the catalytic activity through the blood. The datadescribed herein support the following conclusions: ASPH overexpressioncauses increased motility, migration, invasion and metastasis of HCCcells as well as other human tumor cell lines; many human solid tumorswith a dismal prognosis overexpress ASPH on the cell surface includingbut not limited to pancreatic, hepatocellular, cholangio-, colon,breast, prostate, lung, and glioblastoma cancers; the catalytic site andenzymatic activity are critical for ASPH mediated malignanttransformation, and the subsequent generation of an invasive andmetastatic tumor phenotype; ASPH exerts its biologic effects onincreased migration, invasion, and metastasis of tumor cells byactivation of Notch signaling cascade; small molecule inhibitors of theβ-hydroxylase activity have been discovered based on the crystalstructure of the C-terminal catalytic site of ASPH; tumor cells exposedto these inhibitors such as Compound 310 {8c or MO-I-1100} and Compound405 {5c or MO-I-500} have reduced proliferation, migration, invasion,and colony formation in soft agar which impairs the ability of thesecells to grow and metastasize; these small molecule inhibitors of ASPHenzymatic activity have striking and unexpected anti-tumor effects invivo in animal models of human pancreatic and liver cancer growth anddevelopment. Thus, these studies demonstrate that compounds whichspecifically inhibit the β-hydroxylase activity are useful to reduce thegrowth and/or inhibit metastases of ASPH expressing human solid tumors,in particular those known to have a dismal clinical prognosis, e.g.,Pancreatic Cancer, Hepatocellular Cancer, Cholangiocarcinoma, Lung,Colon Cancer, Breast Cancer, Prostatic Cancer, and Glioblastoma.

In another aspect, this invention features a compound of Formula Ia:

or a salt, ester, metabolite, prodrug, or solvate thereof, wherein

Ar¹ is substituted or unsubstituted C₆-C₂₀ aryl or 5 to 20-memberedheteroaryl;

X is C(O), C(S), or S(O)₂;

W¹ is a single bond, O, CR⁵⁰R⁵¹, or NR⁵² when X is CO and W¹ is a singlebond, CR⁵⁰R⁵¹, or NR⁵² when X is SO₂; and

each of R⁵⁰, R⁵¹, R⁵², and R⁵³ independently is selected from the groupconsisting of hydrogen, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted C₂-C₆ alkenyl, substituted or unsubstitutedC₂-C₆ alkynyl, substituted or unsubstituted C₆-C₂₀ aryl, substituted orunsubstituted C₇-C₂₆ arylalkyl, substituted or unsubstituted 5 to20-membered heteroaryl, and substituted or unsubstituted 6-26 memberedheteroarylalkyl, provided that when Ar¹ is 4-chlorophenyl, then R⁵³ isnot methyl or unsubstituted phenyl.

The compound of Formula Ia may have one or more of the followingfeatures when applicable.

For example, the compound is of Formula IIa:

or a salt, ester, metabolite, prodrug, or solvate thereof, wherein

each of Ar¹ and Ar² independently is unsubstituted C₆-C₁₄ aryl,unsubstituted 5 to 14-membered heteroaryl, or C₆-C₁₄ aryl or 5 to14-membered heteroaryl each substituted with one or more substituentsselected from the group consisting of halo, CN, NO₂, NO, N₃, OR_(a),NR_(a)R_(b), C(O)R_(a), C(O)OR_(a), C(O)NR_(a)R_(b), NR_(b)C(O)R_(a),—S(O)_(b)R_(a), —S(O)_(b)NR_(a)R_(b), or R_(S1), in which R_(S1) isC₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 5- or 6-membered heteroaryl, or 4 to 12-membered heterocycloalkyl,b is 0, 1, or 2, each of R_(a) and R_(b), independently is H or R_(S2),and R_(S2) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or6-membered heteroaryl; and each of R_(S1) and R_(S2), is optionallysubstituted with one or more substituents selected from the groupconsisting of halo, OH, oxo, C(O)OH, C(O)O—C₁-C₆ alkyl, CN, C₁-C₆ alkyl,C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, and 5- or6-membered heteroaryl.

For example, R⁵³ is unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substitutedwith one or more substituents selected from halo, OH, CN, and amino.

For example, X is S(O)₂ and W¹ is CR⁵⁰R⁵¹.

For example, X is S(O)₂ and W¹ is a single bond.

For example, X is C(O) and W¹ is O, or X is C(S) and W¹ is NR⁵².

For example, each of R⁵⁰, R⁵¹, and R⁵² independently is H, unsubstitutedC₁-C₆ alkyl, or C₁-C₆ alkyl substituted with one or more substituentsselected from halo, OH, CN, and amino.

For example, each of Ar¹ and Ar² independently is phenyl, naphthyl, or 5to 10-membered heteroaryl, each of which is optionally substituted withone or more substituents selected from the group consisting of halo, CN,NO₂, NO, N₃, OR_(a), NR_(a)R_(b), C(O)R_(a), C(O)OR_(a),C(O)NR_(a)R_(b), NR_(b)C(O)R_(a), —S(O)_(b)R_(a), —S(O)_(b)NR_(a)R_(b),or R_(S1), in which R_(S1) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5- or 6-membered heteroaryl, or 4 to12-membered heterocycloalkyl, b is 0, 1, or 2, each of R_(a) and R_(b),independently is H or R_(S2), and R_(S2) is C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, or 5- or 6-membered heteroaryl; and each of R_(S1) andR_(S2), is optionally substituted with one or more substituents selectedfrom the group consisting of halo, OH, oxo, C(O)OH, C(O)O—C₁-C₆ alkyl,CN, C₁-C₆ alkyl, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl.

For example, each of Ar¹ and Ar² independently is phenyl, naphthyl, or 5to 10-membered heteroaryl, each of which is optionally substituted withone or more substituents selected from the group consisting of halo, CN,NO₂, NO, N₃, OR_(a), NR_(a)R_(b), C(O)R_(a), C(O)OR_(a), or R_(S1), inwhich R_(S1) is C₁-C₆ alkyl, each of R_(a) and R_(b), independently is Hor R_(S2), and R_(S2) is C₁-C₆ alkyl; and each of R_(S1) and R_(S2), isoptionally substituted with one or more substituents selected from thegroup consisting of halo, OH, C₁-C₆ alkoxyl, amino, mono-C₁-C₆alkylamino, and di-C₁-C₆ alkylamino.

For example, each of Ar¹ and Ar² independently is selected from phenyl,1-naphthyl, 2-naphthyl, 2-furanyl, 2-thiazolyl, 2-pyridyl, 3-pyridyl,4-pyridyl, 2-quinolinyl, 3-quinolinyl, 4-quinolinyl, 2-chlorophenyl,3-chlorophenyl, 4-chlorophenyl, 2-fluorophenyl, 3-fluorophenyl,4-fluorophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl,4-trifluoromethylphenyl, 2-cyanophenyl, 3-cyanophenyl, 4-cyanophenyl,3-carboxymethylphenyl, 2-methoxyphenyl, 3-methoxyphenyl,4-methoxyphenyl, 2,3-dichlorophenyl, 2,4-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl,3,4-difluorophenyl, 3,5-difluorophenyl, 2,3-dimethoxyphenyl,2,4-dimethoxyphenyl, 2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl,3,5-dimethoxyphenyl, 2-chloro-6-fluorophenyl, 3-chloro-4-fluorophenyl,2-chloro-4-fluorophenyl, 4-chloro-3-fluorophenyl,3-chloro-2-fluorophenyl, 2-chloro-5-fluorophenyl,4-chloro-2-fluorophenyl, and 5-chloro-2-fluorophenyl.

For example, the compound of Formula Ia or IIa is an ASPH inhibitorycompound.

Therapeutic Uses of Compositions Comprising Compounds of the Invention

In some aspects, this invention provides for the use of a compound asherein described, or its isomer, metabolite, tautomer, pharmaceuticallyacceptable salt, pharmaceutical product, polymorph, crystal, N-oxide,hydrate, or any combination thereof, for treating, suppressing,preventing, reducing the severity, reducing the risk, or inhibiting acell proliferation disorder in a subject.

Pharmaceutical Compositions

Related aspects of the invention are directed to compositions, includingpharmaceutical compositions, comprising the compounds of the invention,noted above. One aspect of the invention is directed to a pharmaceuticalcomposition comprising at least one pharmaceutically acceptableexcipient and a therapeutically effective amount of the compound or saltdisclosed above. Still another aspect of the invention relates to amethod for pharmaceutical formulation of previously described compoundsfor use in oral and intravenous applications, and in implantablematerials.

Another aspect of the present invention relates to a pharmaceuticalcomposition including a pharmaceutical composition can contain one ormore of the above-identified compounds of the present invention.

Typically, the pharmaceutical composition of the present invention willinclude a compound of the present invention or its pharmaceuticallyacceptable salt, as well as a pharmaceutically acceptable carrier. Theterm “pharmaceutically acceptable carrier” refers to any suitableadjuvants, carriers, excipients, or stabilizers, and can be in solid orliquid form such as, tablets, capsules, powders, solutions, suspensions,emulsions, or implantable disc.

Typically, the composition will contain from about 0.01 to 99 percent,preferably from about 20 to 75 percent of active compound(s), togetherwith the adjuvants, carriers and/or excipients. While individual needsmay vary, determination of optimal ranges of effective amounts of eachcomponent is within the skill of the art. Typical dosages comprise about0.01 to about 100 mg/kg body wt. The preferred dosages comprise about0.1 to about 100 mg/kg body wt. The most preferred dosages compriseabout 1 to about 100 mg/kg body wt. Treatment regimen for theadministration of the compounds of the present invention can also bedetermined readily by those with ordinary skill in art. That is, thefrequency of administration and size of the dose can be established byroutine optimization, preferably while minimizing any side effects.

Dosage Forms

The solid unit dosage forms can be of the conventional type. The solidform can be a capsule and the like, such as an ordinary gelatin typecontaining the compounds of the present invention and a carrier, forexample, lubricants and inert fillers such as, lactose, sucrose, orcornstarch. In another embodiment, these compounds are tabulated withconventional tablet bases such as lactose, sucrose, or cornstarch incombination with binders like acacia, cornstarch, or gelatin,disintegrating agents, such as cornstarch, potato starch, or alginicacid, and a lubricant, like stearic acid or magnesium stearate.

The tablets, capsules, and the like can also contain a binder such asgum tragacanth, acacia, com starch, or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as com starch, potatostarch, alginic acid; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, lactose, or saccharin. When the dosageunit form is a capsule, it can contain, in addition to materials of theabove type, a liquid carrier such as a fatty oil.

Optional Coatings

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets can be coatedwith shellac, sugar, or both. A syrup can contain, in addition to activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye, and flavoring such as cherry or orange flavor.

Excipients

For oral therapeutic administration, these active compounds can beincorporated with excipients and used in the form of tablets, capsules,elixirs, suspensions, syrups, and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compound in these compositions can, of course, bevaried and can conveniently be between about 2% to about 60% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained. Preferred compositions according to the present inventionare prepared so that an oral dosage unit contains between about 1 mg and800 mg of active compound.

Modes of Administration

The active compounds of the present invention may be orallyadministered, for example, with an inert diluent, or with an assailableedible carrier, or they can be enclosed in hard or soft shell capsules,or they can be compressed into tablets, or they can be incorporateddirectly with the food of the diet.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form should be sterile and should befluid to the extent that easy syringability exists. It should be stableunder the conditions of manufacture and storage and should be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (e.g., glycerol, propylene glycol, andliquid polyethylene glycol), suitable mixtures thereof, and vegetableoils.

The compounds or pharmaceutical compositions of the present inventionmay also be administered in injectable dosages by solution or suspensionof these materials in a physiologically acceptable diluent with apharmaceutical adjuvant, carrier or excipient. Such adjuvants, carriersand/or excipients include, but are not limited to, sterile liquids, suchas water and oils, with or without the addition of a surfactant andother pharmaceutically and physiologically acceptable components.Illustrative oils are those of petroleum, animal, vegetable, orsynthetic origin, for example, peanut oil, soybean oil, or mineral oil.In general, water, saline, aqueous dextrose and related sugar solution,and glycols, such as propylene glycol or polyethylene glycol, arepreferred liquid carriers, particularly for injectable solutions.

The pharmaceutical forms suitable for implantable use include sterilewafers of polycarboxyphenoxypropane-sebacic-acid (pCPP:SA) polymers,poly(D,L-lactic acid), polyhydroxybutyrate, lysine diisocyanate(LDI)-glycerol polyurethane, and poly(D-L lactide-co-glycolide). In allcases, the form should be sterile and should be a wafer or disc ofsuitable dimensions for surgical implantation in the brain. The polymersshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganisms,such as bacteria and fungi. The wafers should be biodegradable rangingfrom 24 hours up to 6 months.

In one aspect, the invention provides compounds and compositions,including any aspect described herein, for use in any of the methods ofthis invention. In one aspect, use of a compound of this invention or acomposition comprising the same, will have utility in inhibiting,suppressing, enhancing or stimulating a desired response in a subject,as will be understood by one skilled in the art. In another embodiment,the compositions may further comprise additional active ingredients,whose activity is useful for the particular application for which thecompound of this invention is being administered.

Pharmaceutical Compositions comprising Modulator Compounds of theInvention

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, monostearate salts and gelatin. Moreover, the modulators can beadministered in a time release formulation, for example in a compositionwhich includes a slow release polymer. The active compounds can beprepared with carriers that will protect the compound against rapidrelease, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, polylactic acid andpolylactic, polyglycolic copolymers (PLG). Many methods for thepreparation of such formulations are patented or generally known tothose skilled in the art.

The mode of administration may be oral, for intestinal delivery;intranasal, for nasal delivery; and intravenous for delivery through theblood-brain barrier. Other modes of administration as are known in theart may also be used, including, but not limited to intrathecal,intramuscular, intrabronchial, intrarectal, intraocular, andintravaginal delivery.

The modulator compounds can be administered as oral dosage compositionsfor small intestinal delivery. Such oral dosage compositions for smallintestinal delivery are well-known in the art, and generally comprisegastroresistent tablets or capsules (Remington's PharmaceuticalSciences, 16th Ed., Eds. Osol, Mack Publishing Co., Chapter 89 (1980);Digenis et al, J. Pharm. Sci., 83:915-921 (1994); Vantini et al, ClinicaTerapeutica, 145:445-451 (1993); Yoshitomi et al, Chem. Pharm. Bull.,40:1902-1905 (1992); Thoma et al, Pharmazie, 46:331-336 (1991);Morishita et al, Drug Design and Delivery, 7:309-319 (1991); and Lin etal, Pharmaceutical Res., 8:919-924 (1991)); each of which isincorporated by reference herein in its entirety).

Tablets are made gastroresistent by the addition of compounds such ascellulose acetate phthalate or cellulose acetate terephthalate.

Capsules are solid dosage forms in which the tight junction modulatorcompound is enclosed in either a hard or soft, soluble container orshell of gelatin. The gelatin used in the manufacture of capsules isobtained from collagenous material by hydrolysis. There are two types ofgelatin. Type A, derived from pork skins by acid processing, and Type B,obtained from bones and animal skins by alkaline processing. The use ofhard gelatin capsules permit a choice in prescribing a tight junctionmodulator compound or a combination thereof at the exact dosage levelconsidered best for the individual subject. The hard gelatin capsuleconsists of two sections, one slipping over the other, thus completelysurrounding the tight junction modulator compound. These capsules arefilled by introducing the modulator compound, or gastroresistent beadscontaining the modulator compound, into the longer end of the capsule,and then slipping on the cap. Hard gelatin capsules are made largelyfrom gelatin, FD&C colorants, and sometimes an opacifying agent, such astitanium dioxide. The USP permits the gelatin for this purpose tocontain 0.15% (w/v) sulfur dioxide to prevent decomposition duringmanufacture.

In the context of the present invention, oral dosage compositions forsmall intestinal delivery also include liquid compositions which containaqueous buffering agents that prevent the modulator compound from beingsignificantly inactivated by gastric fluids in the stomach, therebyallowing the modulator compound to reach the small intestines in anactive form. Examples of such aqueous buffering agents which can beemployed in the present invention include bicarbonate buffer (pH 5.5 to8.7, preferably about pH 7.4).

When the oral dosage composition is a liquid composition, it ispreferable that the composition be prepared just prior to administrationso as to minimize stability problems. In this case, the liquidcomposition can be prepared by dissolving lyophilized tight junctionmodulator compound in the aqueous buffering agent. Oral dosagecompositions for small intestinal delivery also include liquidcompositions which may optionally contain aqueous buffering agents thatprevent the therapeutic agent and tight junction modulator compound frombeing significantly inactivated by gastric fluids in the stomach,thereby allowing the biologically active ingredient and tight junctionmodulator compound to reach the small intestines in an active form.Examples of such aqueous buffering agents which can be employed in thepresent invention include bicarbonate buffer (pH 5.5 to 8.7, preferablyabout pH 7.4).

When the oral dosage composition is a liquid composition, it ispreferable that the composition be prepared just prior to administrationso as to minimize stability problems. In this case, the liquidcomposition can be prepared by dissolving lyophilized therapeutic agentand tight junction modulator compound in the aqueous buffering agent.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. For sterile powders used in the preparation of sterileinjectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

A “nasal” delivery composition differs from an “intestinal” deliverycomposition in that the latter must have gastroresistent properties inorder to prevent the acidic degradation of the active agents in thestomach, whereas the former generally comprises water-soluble polymerswith a diameter of about 50 11 m in order to reduce the mucociliaryclearance, and to achieve a reproducible bioavailability of the nasallyadministered agents.

An “intravenous” delivery composition differs from both the “nasal” and“intestinal” delivery compositions in that there is no need forgastroresistance or water-soluble polymers in the “intravenous” deliverycomposition.

Nasal dosage compositions for nasal delivery are well-known in the art.Such nasal dosage compositions generally comprise water-soluble polymersthat have been used extensively to prepare pharmaceutical dosage forms(Martin et al, In: Physical Chemical Principles of 20 PharmaceuticalSciences, 3rd Ed., pages 592-638 (1983)) that can serve as carriers forpeptides for nasal administration (Davis, In: Delivery Systems forPeptide Drugs, 125:1-21 (1986)). The nasal absorption of pap tidesembedded in polymer matrices has been shown to be enhanced throughretardation of nasal mucociliary clearance (Illum et al, Int. J. Pharm.,46:261-265 (1988É). Other possible enhancement mechanisms include anincreased concentration gradient or 25 decreased diffusion path forpeptides absorption (Ting et al, Pharm. Res., 9:1330-1335 (1992).However, reduction in mucociliary clearance rate has been predicted tobe a good approach toward achievement or reproducible bioavailability ofnasally administered systemic drugs (Gonda et al, Pharm. Res., 7:69-75(1990)). Microparticles with a diameter of about 50 pm are expected todeposit in the nasal cavity (Bjork et al, Int. J. Pharm., 62:187-192(1990); and lllum et al, Int. J. Pharm., 39:189-199 (1987), whilemicroparticles with a diameter under 10 pm can escape the filteringsystem of the nose and deposit in the lower airways. Microparticleslarger than 200 pm in diameter will not be retained in the nose afternasal administration (Lewis et al, Proc. Int. Symp. Control ReI. Bioact.Mater., 17:280-290 (1990)).

The particular water-soluble polymer employed is not critical to thepresent invention, and can be selected from any of the well-knownwater-soluble polymers employed for nasal dosage forms. A typicalexample of a water-soluble polymer useful for nasal delivery ispolyvinyl alcohol (pvA). This material is a swellable hydrophilicpolymer whose physical properties depend on the molecular weight, degreeof hydrolysis, cross-linking density, and crystallinity (Peppas et al,In: Hydrogels in Medicine and Pharmacy, 3:109-131 (1987). PYA can beused in the coating of dispersed materials through phase separation,spray-drying, spray-embedding, and spray-densation (Ting et al, supra).

A “skin” delivery composition comprising a modulator compound of theinvention may include in addition a therapeutic or immunogenic agent,fragrance, creams, ointments, colorings, and other compounds so long asthe added component does not deleteriously affect transdermal deliveryof the therapeutic or immunogenic agent. Conventional pharmaceuticallyacceptable emulsifiers, surfactants, suspending agents, antioxidants,osmotic enhancers, extenders, diluents and preservatives may also beadded. Water soluble polymers can also be used as carriers.

The particular therapeutic or immunogenic agent employed is not criticalto the present invention, and can be, e.g., any drug compound,biologically active peptide, vaccine, or any other moiety otherwise notabsorbed through the transcellular pathway, regardless of size orcharge.

The amount of active compound in the composition may vary according tofactors such as the disease state, age, sex, and weight of theindividual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically 35 discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

As used herein “pharmaceutically-acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. In one embodiment, the carrier is 10suitable for parenteral administration. A carrier may be suitable foradministration into the central nervous system (e.g., intraspinally orintracerebrally). Alternatively, the carrier can be suitable forintravenous, intraperitoneal or intramuscular administration. In anotherembodiment, the carrier is suitable for oral administration.Pharmaceutically-acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

While specific aspects of the invention have been described in detail,it will be appreciated by those skilled in the art that variousmodifications and alternatives to those details could be developed inlight of the overall teachings of the disclosure. Accordingly, theparticular arrangements disclosed are meant to be illustrative only, andnot limiting as to the scope of the invention, which is to be given thefull breadth of the appended claims, and any equivalent, thereof.

EXAMPLES

The foregoing discussion may be better understood in connection with thefollowing representative examples which are presented for purposes ofillustrating the principle methods and compositions of the invention,and not by way of limitation. Various other examples will be apparent tothe person skilled in the art after reading the present disclosurewithout departing from the spirit and scope of the invention. It isintended that all such other examples be included within the scope ofthe appended claims.

General Materials and Methods

All parts are by weight (e.g., % w/w), and temperatures are in degreescentigrade (° C.), unless otherwise indicated.

General Chemical Procedures

Melting points were determined with a Hoover melting point apparatus andare uncorrected. Infrared (IR) spectra for the compounds were recordedin KBr discs on a Mattson Satellite FTIR in cm⁻¹. ¹H and ¹³C spectrawere recorded in DMSO-d₆ on a Bruker Avance III DPX 300 MHz instrument.¹⁹F spectra were recorded in DMSO d₆ on a Bruker Avance III 600 (564.6mHz). Chemical shifts were expressed in parts per million (δ) withtetramethylsilane as internal standard. Mass spectrometry was performedon a Thermo Scientific LTQ-FT at the University of Cincinnati MassSpectrometry facility. The purity of the compounds was monitored by HPLCusing a Waters 2695 separation module and a 2487 dual λ absorbancedetector with a NovaPak C18 4 μm 3.9×150 mm column. The mobile phasesconsisted of acetonitrile/H₂O using a 30 minute gradient. All compoundswere ≧95%. Microanalysis was performed by Atlantic Microlab Inc., andall compounds were found to be ±0.4%. All reagents were fromSigma-Aldrich. LogS, LogP, Log BBB, human intestinal absorption,p-glycoprotein category, CYP 2C9 pKi, hERG pIC50, CYP 2D6 affinitycategory, oral CNS score, IV CNS score, MW, flexibility, and total polarsurface area were calculated using StarDrop 5.1.1 release Build 178.

Scheme 1 illustrates the synthetic reactions used to summarize thesereactions. Table 2 is a non-limiting list of aryl functional groups thatcan be incorporated as “Ar¹” or “Ar²” from Formulae Ia, Ib, and IIa.Tables 3 and 4 illustrate the structures, names, and numbers of avariety of key compounds disclosed in in this application.

TABLE 2 A Non-Limiting List of Aryl Functional Groups That Can BeIncorporated as “Ar¹” or “Ar²” From Formulae Ia, Ib, and IIa Aryl Func-Old Aryl tional Functional Functional Group # Group # Group StructureAr¹ or Ar² 201 a

2-chlorophenyl 202 b

3-chlorophenyl 203 c

4-chlorophenyl 204 d

2,3-dichlorophenyl 205 e

2,4-dichlorophenyl 206 f

2,5-dichlorophenyl 207 g

3- carboxymethylphenyl 208 h

3,4-dichlorophenyl 209 i

3,5-dichlorophenyl 210 j

2-fluorophenyl 211 k

3-fluorophenyl 212 l

4-fluorophenyl 213 m

2,3-difluorophenyl 214 n

2,4-difluorophenyl 215 o

2,5-difluorophenyl 216 p

2,6-difluorophenyl 217 q

3,4-difluorophenyl 218 r

3,5-difluorophenyl 219 s

2-methoxyphenyl 220 t

3-methoxyphenyl 221 u

4-methoxyphenyl 222 v

2,3- dimethoxyphenyl 223 w

2,4- dimethoxyphenyl 224 x

2,5- dimethoxyphenyl 225 y

2,6- dimethoxyphenyl 226 z

3,4- dimethoxyphenyl 227 aa

3-5- dimethoxyphenyl 228 ab

2-chloro-6- fluorophenyl 229 ac

3-chloro-4- fluorophenyl 230 ad

2-chloro-4- fluorophenyl 231 ae

4-chloro-3- fluorophenyl 232 af

3-chloro-2- fluorophenyl 233 ag

2-chloro-5- fluorophenyl 234 ah

4-chloro-2- fluorophenyl 235 ai

5-chloro-2- fluorophenyl 236 aj

Ph 237 ak

2-thiophene 238 al

2-furan 239 am

2-thiazole 240 an

1-naphthyl 241 ao

2-napthyl 242 ap

2-pyridyl 243 aq

3-pyridyl 244 ar

4-pyridyl 245 as

2-quinolinyl 246 at

3-quinolinyl 247 au

4-quinolinyl 248 av

4- trifluoromethylphenyl 249 aw

3- trifluoromethylphenyl 250 ax

2- trifluoromethylphenyl 251 ay

4-cyanophenyl 252 az

3-cyanophenyl

TABLE 3 Structures, Names, and Numbers of a Variety of Key CompoundsListed in Example I New Cpd # Old Cpd # Structure Name 301 1c

2-(4-chlorophenyl)-4- [[methylsulfonyl]oxy]-5- amino-3(2H)-furanone 3022c

2-(4-chlorophenyl)-4- [[ethylsulfonyl]oxy]-5- amino-3(2H)-furanone 3033c

2-(4-chlorophenyl)-4- [[1-propylsulfonyl]oxy]- 5-amino-3(2H)-furanone304 4c

2-(4-chlorophenyl)-4- [[2-propylsulfonyl]oxy]- 5-amino-3(2H)-furanone305 5c

2-(4-chlorophenyl)-4- [[1-butylsulfonyl]oxy]- 5-amino-3(2H)-furanone 3066c

2-(4-chlorophenyl)-4- [[1-propyl-2-methyl- sulfonyl]oxy]-5-amino-3(2H)-furanone 307 7c, MO- I-1000

2-(4-chlorophenyl)-4- [[phenylsulfonyl]oxy]- 5-amino-3(2H)-furanone 3088a

2-(2-chlorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 309 8b

2-(3-chlorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 310 8c, MO- I-1100

2-(4-chlorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 311 8d

2-(2,3-dichlorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 312 8e

2-(2,4-dichlorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 313 8f

2-(2,5-dichlorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 314 8g, MO- I-1150

2-(3-carboxymethylphenyl)- 4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 315 8h, MO- I-1144

2-(3,4-dichlorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 317 8j

2-(2-fluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 318 8k

2-(3-fluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 319 8l

2-(4-fluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 320 8m

2-(2,3-difluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 321 8n

2-(2,4-difluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 322 8o

2-(2,5-difluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 323 8p

2-(2,6-difluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 324 8q

2-(3,4-difluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 325 8r

2-(3,5-difluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 326 8s

2-(2-methoxyphenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 327 8t

2-(3-methoxyphenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 328 8u

2-(4-methoxyphenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 329 8v

2-(2,3-dimethoxyphenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 330 8w

2-(2,4-dimethoxyphenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 331 8x

2-(2,5-dimethoxyphenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 332 8y

2-(2,6-dimethoxyphenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 333 8z

2-(3,4-dimethoxyphenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 334 8aa

2-(3,5-dimethoxyphenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 335 8ab

2-(2-chloro-6-fluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 336 8ac

2-(3-chloro-4-fluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 337 8ad

2-(2-chloro-4-fluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 338 8ae

2-(4-chloro-3-fluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 339 8af

2-(3-chloro-2-fluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 340 8ag

2-(2-chloro-5-fluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 341 8ah

2-(4-chloro-2-fluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 342 8ai

2-(3-chloro-5-fluorophenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 343 8aj

2-(phenyl)-4- [[phenylmethylsulfonyl]oxy]- 5-amino-3(2H)-furanone 3448ak

2-(2-thiophene)-4- [[phenylmethylsulfonyl]oxy]- 5-amino-3(2H)-furanone345 8al

2-(2-furanyl)-4- [[phenylmethylsulfonyl]oxy]- 5-amino-3(2H)-furanone 3468am

2-(2-thiazolyl)-4- [[phenylmethylsulfonyl]oxy]- 5-amino-3(2H)-furanone347 8an

2-(1-naphthyl)-4- [[phenylmethylsulfonyl]oxy]- 5-amino-3(2H)-furanone348 8ao

2-(2-naphthyl)-4- [[phenylmethylsulfonyl]oxy]- 5-amino-3(2H)-furanone349 8ap

2-(2-pyridyl)-4- [[phenylmethylsulfonyl]oxy]- 5-amino-3(2H)-furanone 3508aq

2-(3-pyridyl)-4- [[phenylmethylsulfonyl]oxy]- 5-amino-3(2H)-furanone 3518ar

2-(4-pyridyl)-4- [[phenylmethylsulfonyl]oxy]- 5-amino-3(2H)-furanone 3528as

2-(2-quinolinyl)-4- [[phenylmethylsulfonyl]oxy]- 5-amino-3(2H)-furanone353 8at

2-(3-quinolinyl)-4- [[phenylmethylsulfonyl]oxy]- 5-amino-3(2H)-furanone354 8au

2-(4-quinolinyl)-4- [[phenylmethylsulfonyl]oxy]- 5-amino-3(2H)-furanone355 8av, MO- I-1151

2-(4-trifluoromethylphenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 356 8aw

2-(3-trifluoromethylphenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 357 8ax

2-(2-trifluoromethylphenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 358 8ay

2-(4-nitrilephenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 359 8az

2-(3-nitrilephenyl)-4- [[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 360 9c

2-(4-chlorophenyl)-4- [[1-phenylethylsulfonyl]oxy]-5-amino-3(2H)-furanone 361 10c

2-(4-chlorophenyl)-4- [[1-methyl-1- phenylethylsulfonyl]oxy]-5-amino-3(2H)-furanone 362 11c

2-(4-chlorophenyl)-4- [[4-methylphenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 363 12c

2-(4-chlorophenyl)-4- [3-methylphenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 364 13c

2-(4-chlorophenyl)-4- [[2-methylphenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 365 14c

2-(4-chlorophenyl)-4- [[4-chlorophenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 366 15c

2-(4-chlorophenyl)-4- [[3-chlorophenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 367 16c

2-(4-chlorophenyl)-4- [[2-chlorophenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 368 17c

2-(4-chlorophenyl)-4- [[4-fluorophenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 369 18c

2-(4-chlorophenyl)-4- [[3-fluorophenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 370 19c

2-(4-chlorophenyl)-4- [[2-fluorophenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 371 20c

2-(4-chlorophenyl)-4-[[4- trifluoromethylphenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 372 21c

2-(4-chlorophenyl)-4-[[3- trifluoromethylphenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 373 22c

2-(4-chlorophenyl)-4-[[2- trifluoromethylphenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 374 23c

2-(4-chlorophenyl)-4- [[4-pyridylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 375 24c

2-(4-chlorophenyl)-4- [[3-pyridylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 376 25c

2-(4-chlorophenyl)-4- [[2-pyridylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 377 26c

2-(4-chlorophenyl)-4-[[3,4- difluorophenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 378 27c

2-(4-chlorophenyl)-4-[[2,3- difluorophenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 379 28c

2-(4-chlorophenyl)-4-[[2,4- difluorophenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 380 29c

2-(4-chlorophenyl)-4-[[3,5- difluorophenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 381 30c

2-(4-chlorophenyl)-4-[[2,5- difluorophenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 382 31c

2-(4-chlorophenyl)-4-[[2,6- difluorophenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 383 32c

2-(4-chlorophenyl)-4-[[1- naphthylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 384 33c

2-(4-chlorophenyl)-4- [[2-naphthylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone 385 34c

2-(4-chlorophenyl)-4- [[2-thiophenemethylsulfonyl]oxy]-5-amino-3(2H)-furanone 386 35c

2-(4-chlorophenyl)-4- [[phenoxycarbonyl]oxy]-5- amino-3(2H)-furanone 38736c

2-(4-chlorophenyl)-4- [[benzyloxycarbonyl]oxy]-5- amino-3(2H)-furanone388 37c

2-(4-chlorophenyl)-4- [[phenylaminothiocarbonyl]oxy]-5-amino-3(2H)-furanone 389 38c

2-(4-chlorophenyl)-4- [[benzylaminothiocarbonyl]oxy]-5-amino-3(2H)-furanone

TABLE 4 Structures, Names, and Numbers of a Variety of Key CompoundsListed in Example 2 New Cpd # Old Cpd # Structure Name 401 1c, MO-I-100

5-amino-4-hydroxy-2- (4-chlorophenyl)-furan-3-one 402 2c, MO-I-300

2-(4-chlorophenyl)-4-(acetoxy)- 5-amino-3(2H)-furanone 403 3c,MO-I-1000, identical to 307

2-(4-chlorophenyl)-4- [[phenylsulfonyl]oxy]-5- amino-3(2H)-furanone 4044c, MO-I-400

N-(3,4-dihydroxy-5- (4-chlorophenyl)-2-furanyl) methanesulfonamide 4055c, MO-I-500

N-(3,4-dihydroxy-5- (4-chlorophenyl)-2-furanyl) ethanesulfonamide 406 6c

N-(3,4-dihydroxy-5- (4-chlorophenyl)-2-furanyl) benzenesulfonamide 4077c, MO-I-1600 series

N-(3,4-dihydroxy-5- (4-chlorophenyl)-2- furanyl)acetamide 408 8c,MO-I-1600 series

N-(3,4-dihydroxy-5- (4-chlorophenyl)-2-furanyl)- carbamic acid ethylester 409 9c

N-(3,4-dihydroxy-5- (4-chlorophenyl)-2- furanyl)benzamide 410 10c

N-(3-trimethylsilyloxy-4- dihydroxy-5-(4-chlorophenyl)-2-furanyl)-4-bromo-butanamide 411 11c

N-(3,4-dihydroxy-5- (4-chlorophenyl)-2-furanyl)- 2-pyrrolidinone 412 12c

N-(3,4-dihydroxy-5- (4-chlorophenyl)-2-furanyl) succinimide 413 13c

N-(3-trimethylsilyloxy-4- dihydroxy-5-(4-chlorophenyl)-2-furanyl)-3-bromo- propylsulfonamide 414 14c

N-(3,4-dihydroxy-5- (4-chlorophenyl)-2-furanyl)-1,1-dioxide-isothiazolidine 415 15c

N-methyl-N'-(3,4-dihydroxy-5- (4-chlorophenyl)-2-furanyl)ethanesulfonamide 416 16c

N-(3-acetoxy-4-hydroxy-5- (4-chlorophenyl)-2-furanyl) methanesulfonamide417 17c

N-(3-acetoxy-4-hydroxy-5- (4-chlorophenyl)-2-furanyl) ethanesulfonamide418 18c

N-(3-acetoxy-4-hydroxy-5- (4-chlorophenyl)-2-furanyl) benzenesulfonamide419 19c

N-(3-acetoxy-4-hydroxy-5- (4-chlorophenyl)-2-furanyl)- acetamide 420 20c

N-(3-acetoxy-4-hydroxy-5- (4-chlorophenyl)-2-furanyl)- carbamic acidethyl ester 421 21c

N-(3-acetoxy-4-hydroxy-5- (4-chlorophenyl)-2-furanyl) benzamide 422 22c

N-(3-acetoxy-4-hydroxy-5- (4-chlorophenyl)-2-furanyl)-4-bromo-butanamide 423 23c

N-(3-acetoxy-4-hydroxy-5- (4-chlorophenyl)-2-furanyl)-2- pyrrolidinone424 24c

N-(3-acetoxy-4-hydroxy-5- (4-chlorophenyl)-2-furanyl) succinimide 42525c

N-(3-acetoxy-4-hydroxy-5- (4-chlorophenyl)-2-furanyl)-3-bromo-propylsulfonamide 426 26c

N-(3-acetoxy-4-hydroxy-5- (4-chlorophenyl)-2-furanyl)-1,1-dioxide-isothiazolidine 427 27c

N-methyl-N′-(3-acetoxy-4- hydroxy-5-(4-chlorophenyl)-2-furanyl)ethanesulfonamide 428 28c

5-amino-2-(4-chlorophenyl)- 2-methyl-4-trimethylsilyloxy- 3(2H)-furanone429 29c

5-amino-2-(4-chlorophenyl)- 2-methyl-4-hydroxy-3(2H)- furanone 430 30c

N-(3-hydroxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-methanesulfonamide 431 31c

N-(3-hydroxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-ethanesulfonamide 432 32c

N-(3-hydroxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-benzenesulfonamide 433 33c

N-(3-hydroxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-acetamide434 34c

N-(3-hydroxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)- carbamicacid ethyl ester 435 35c

N-(3-hydroxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-benzamide436 36c

N-(3-hydroxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-4-bromo-butanamide 437 37c

N-(3-hydroxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-2-pyrrolidinone 438 38c

N-(3-hydroxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-succinimide 439 39c

N-(3,4-dihydroxy-5-(4-chlorophenyl)-2-furanyl)-3-bromo-propylsulfonamide 440 40c

N-(3-hydroxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-1,1-dioxide-isothiazolidine 441 41c, MO-I-500 series

N-methyl-N′-(3-hydroxy-5- (4-chlorophenyl)-5-methyl-2- 3(2H)-furanonyl)-ethanesulfonamide 442 42c, MO-I-300 series

5-amino-2-(4-chlorophenyl)- 2-methyl-4-acetoxy-3(2H)- furanone 443 43c

N-(3-acetoxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-methanesulfonamide 444 44c

N-(3-acetoxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-ethanesulfonamide 445 45c

N-(3-acetoxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-benzenesulfonamide 446 46c

N-(3-acetoxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)- acetamide447 47c

N-(3-acetoxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)- carbamicacid ethyl ester 448 48c

N-(3-acetoxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-benzamide449 49c

N-(3-acetoxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-4-bromo-butanamide 450 50c

N-(3-acetoxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-2-pyrrolidinone 451 51c

N-(3-acetoxy-5-(4-clorophenyl)- 5-methyl-2-3(2H)-furanonyl)- succinimide452 52c

N-(3-acetoxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-3-bromo-propylsulfonamide 453 53c

N-(3-acetoxy-5-(4-chlorophenyl)- 5-methyl-2-3(2H)-furanonyl)-1,1-dioxide-isothiazolidine 454 54c

N-methyl-N′-(3-acetoxy-5- (4-chlorophenyl)-5-methyl- 2-3(2H)-furanonyl)-ethanesulfonamide 455 1j

5-amino-4-hydroxy-2- (2-fluorophenyl)-furan-3-one 456 1k

5-amino-4-hydroxy-2- (3-fluorophenyl)-furan-3-one 457 1l

5-amino-4-hydroxy-2- (4-fluorophenyl)-furan-3-one 458 1m

5-amino-4-hydroxy-2- (2,3-difluorophenyl)-furan-3-one 459 1n

5-amino-4-hydroxy-2- (2,4-difluorophenyl)-furan-3-one 460 1o

5-amino-4-hydroxy-2- (2,5-difluorophenyl)-furan-3-one 461 1p

5-amino-4-hydroxy-2- (2,6-difluorophenyl)-furan-3-one 462 1q

5-amino-4-hydroxy-2- (3,4-difluorophenyl)-furan-3-one 463 1r

5-amino-4-hydroxy-2- (3,5-difluorophenyl)-furan-3-one

Example 1 Synthesis and Characterization of Compounds in Table 3 GeneralProcedures for Preparation of Aryltetronimides

Potassium cyanide (0.91 g) was added to sodium carbonate (1.7 g) indeionized water (30 mL) in a 3-Neck Glass Round Flask and placed in anice bath. The system was repeatedly purged using a vacuum pump andnitrogen gas. Glyoxal (3.72 g) was then added to the system without theintroduction of O₂ and the reactants were allowed to dissolve withstirring. In a stoppered tube, the appropriate arylaldehyde (7.11mmoles) was added to 1,4-dioxane (5 mL), purged, and then addeddrop-wise to the system. The system was then removed from the ice bathand allowed to stir at room temperature for 1 hour. After 1 hour, aceticacid (5 mL) was added drop-wise until gas bubbles were no longer visiblefrom the addition of acetic acid, or until the solution was at a pH ofless than 6. The solution was vacuum filtered and washed with ice coldwater (5 mL), methanol (5 mL) and ether (5 mL) and then was allowed toair dry. Crude material was recrystallized with methanol, collected byvacuum filtration and rinsed with diethyl ether and dried under vacuum.

Compound 301{1c}:2-(4-chlorophenyl)-4-[[methylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-onein dry THF under argon is added triethylamine in dry THF. The reactionis stirred at room temperature for 30 minutes, and 1 eq ofmethanesulfonyl chloride in dry THF is added dropwise. The reaction isstirred for 16 hours, followed by the addition of a saturated ammoniumchloride solution. The mixture is extracted 3 times with 30 mL ofdiethyl ether. The combined ether extracts are washed with water andbrine, dried over sodium sulfate, filtered, and concentrated underreduced pressure. The solid is recrystallized with methanol.

Compound 301{2c}:2-(4-chlorophenyl)-4-[[ethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-onein dry THF under argon is added triethylamine in dry THF. The reactionis stirred at room temperature for 30 minutes, and 1 eq ofethanesulfonyl chloride in dry THF is added dropwise. The reaction isstirred for 16 hours, followed by the addition of a saturated ammoniumchloride solution. The mixture is extracted 3 times with 30 mL ofdiethyl ether. The combined ether extracts are washed with water andbrine, dried over sodium sulfate, filtered, and concentrated underreduced pressure. The solid is recrystallized with methanol.

Compound 303{3c}:2-(4-chlorophenyl)-4-[[1-propylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-onein dry THF under argon is added triethylamine in dry THF. The reactionis stirring at room temperature for 30 minutes, and 1 eq ofn-propanesulfonyl chloride in dry THF is added dropwise. The reaction isstirred for 16 hours, followed by the addition of a saturated ammoniumchloride solution. The mixture is extracted 3 times with 30 mL ofdiethyl ether, the combined ether extracts are washed with water andbrine, dried over sodium sulfate, filtered, and concentrated underreduced pressure. The solid is recrystallized with methanol.

Compound 304{4c}:2-(4-chlorophenyl)-4-[[2-propylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-onein dry THF under argon is added triethylamine in dry THF. The reactionis stirred at room temperature for 30 minutes, and 1 eq ofi-propanesulfonyl chloride in dry THF is added dropwise. The reaction isstirred for 16 hours, followed by the addition of a saturated ammoniumchloride . The mixture is extracted 3 times with 30 mL of diethyl ether.The combined ether extracts are washed with water and brine, dried oversodium sulfate, filtered, and concentrated under reduced pressure. Thesolid is recrystallized with methanol.

Compound 305 {5c}:2-(4-chlorophenyl)-4-[[1-butylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-onein dry THF under argon is added triethylamine in dry THF. The reactionis stirred at room temperature for 30 minutes, and 1 eq ofn-butanesulfonyl chloride in dry THF is added dropwise. The reaction isstirred for 16 hours, followed by the addition of a saturated ammoniumchloride . The mixture is extracted 3 times with 30 mL of diethyl ether,the combined ether extracts are washed with water and brine, dried oversodium sulfate, filtered, and concentrated under reduced pressure. Thesolid is recrystallized with methanol.

Compound 306 {6c}:2-(4-chlorophenyl)-4-[[1-propyl-2-methyl-sulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-onein dry THF under argon is added triethylamine in dry THF. The reactionis stirring at room temperature for 30 minutes, and 1 eq ofi-butanesulfonyl chloride in dry THF is added dropwise. The reaction isstirred for 16 hours, followed by the addition of a saturated ammoniumchloride solution. The mixture is extracted 3 times with 30 mL ofdiethyl ether. The combined ether extracts are washed with water andbrine, dried over sodium sulfate, filtered, and concentrated underreduced pressure. The solid is recrystallized with methanol.

Compound 307 {7c orMO-I-1000}:2-(4-chlorophenyl)-4-[[phenylsulfonyl]oxy]-5-amino-3(2H)-furanone

5 g of 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-one was stirred in50 mL of dry THF under argon gas for 16 hours with 4.7 g K₂CO₃ and 4.25mL of benzenesulfonyl chloride. The reaction was filtered, and thefiltrate was acidified with 24 mL 1N HCl, and extracted 5 times with 20mL of diethyl ether. The combined ether extracts were washed with brineand dried with Na₂SO₄. After filtration, 100 mL of hexanes was added tothe solution, resulting in a precipitate, which was collected usingvacuum filtration and recrystallized with MeOH. Yield=17%. mp 190-195°C.; FTIR 3099, 1630; ¹H NMR (300 MHz, DMSO-d₆, ppm) δ 8.54 (s, 2H), 7.93(d, J=8.1 Hz, 2H), 7.74 (t, J=7.2 Hz, 1H), 7.57 (t, J=8.1 Hz, 2H), 7.48(d, J=8.7 Hz, 2H), 7.18 (d, J=8.4 Hz, 2H), 5.54 (s, 1H). ¹³C NMR (75MHz, DMSO-d₆, ppm) δ 181.2, 173.4, 135.2, 135.0, 134.2, 133.9, 129.7,129.2, 129.1, 129.0, 106.6, 82.9. Elemental Analysis Calc: C 52.54, H3.31, N 3.83, Cl 9.69; Found: C 52.50, H 3.33, N 3.79, Cl 9.84;C₁₆H₁₂ClNO₅S; HPLC retention time: 32.2 min.

Compound 308{8a}:2-(2-chlorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of 5-amino-4-hydroxy-2-(2-chlorophenyl)-furan-3-onein dry THF under argon is added triethylamine in dry THF. The reactionis stirred at room temperature for 30 minutes, and 1 eq ofphenylmethylsulfonyl chloride in dry THF is added dropwise. The reactionis stirred for 16 hours followed by the addition of a saturated ammoniumchloride solution. The mixture is extracted 3 times with 30 mL ofdiethyl ether. The combined ether extracts are washed with water andbrine, dried over sodium sulfate, filtered, and concentrated underreduced pressure. The solid is recrystallized with methanol.

Compound 309{8b}:2-(3-chlorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of 5-amino-4-hydroxy-2-(3-chlorophenyl)-furan-3-onein dry THF under argon is added triethylamine in dry THF. The reactionis stirred at room temperature for 30 minutes, and 1 eq ofphenylmethylsulfonyl chloride in dry THF is added dropwise. The reactionis stirred for 16 hours followed by the addition of a saturated ammoniumchloride solution. The mixture is extracted 3 times with 30 mL ofdiethyl ether, the combined ether extracts are washed with water andbrine, dried over sodium sulfate, filtered, and concentrated underreduced pressure. The solid is recrystallized with methanol.

Compound 310{8c or MO-I-1100}:2-(4-chlorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

2.5 g of 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-one was stirred in100 mL of dry THF. 1.31 mL of TEA was added, and 30 minutes later 2.11 gof phenylmethylsulfonylchloride was added to the reaction. The reactionwas stirred for 24 hours. The reaction was filtered, and the filtratewas acidified with 24 mL 1N HCl, and extracted 5 times with 20 mL ofdiethyl ether. The combined ether extracts were washed with brine, anddried with Na₂SO₄. After filtration, the solution was concentrated underreduced pressure, and the resulting solid was recrystallized frommethanol. Yield=27%. FTIR 2957, 1636; ¹H NMR (300 MHz, DMSO-d₆, ppm) δ8.79 (s, 2H), 7.60-7.49 (m, 4H), 7.43-7.35 (m, 5H), 5.80 (s, 1H), 4.97(d, J=14.1, 1H), 4.90 (d, J=14.1, 1H). ¹³C NMR (75 MHz, DMSO-d₆, ppm) δ181.8, 173.9, 134.3, 134.1, 131.5, 129.3, 129.1, 129.1, 129.0, 128.9,107.9, 83.1, 57.8. Elemental Analysis Calc: C 53.76, H 3.72, N 3.69;Found: C 53.90, H 3.68, N 3.70; C₁₇H₁₄ClNO₅S; HPLC retention time: 32.2min.

Compound 311{8d}:2-(2,3-dichlorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(2,3-dichlorophenyl)-furan-3-one in dry THF underargon is added triethylamine in dry THF. The reaction is stirred at roomtemperature for 30 minutes, and 1 eq of phenylmethylsulfonyl chloride indry THF is added dropwise. The reaction is stirred for 16 hours followedby the addition of a saturated ammonium chloride solution. The mixtureis extracted 3 times with 30 mL of diethyl ether. The combined etherextracts are washed with water and brine, dried over sodium sulfate,filtered, and concentrated under reduced pressure. The solid isrecrystallized with methanol.

Compound 312 {8e}:2-(2,4-dichlorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(2,4-dichlorophenyl)-furan-3-one in dry THF underargon is added triethylamine in dry THF. The reaction is stirred at roomtemperature for 30 minutes, and 1 eq of phenylmethylsulfonyl chloride indry THF is added dropwise. The reaction is stirred for 16 hours,followed by the addition of a saturated ammonium chloride solution. Themixture is extracted 3 times with 30 mL of diethyl ether. The combinedether extracts are washed with water and brine, dried over sodiumsulfate, filtered, and concentrated under reduced pressure. The solid isrecrystallized with methanol.

Compound 313{8f}:2-(2,5-dichlorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(2,5-dichlorophenyl)-furan-3-one in dry THF underargon is added triethylamine in dry THF. The reaction is stirred at roomtemperature for 30 minutes, and 1 eq of phenylmethylsulfonyl chloride indry THF is added dropwise. The reaction is stirred for 16 hours followedby the addition of a saturated ammonium chloride solution. The mixtureis extracted 3 times with 30 mL of diethyl ether. The combined etherextracts are washed with water and brine, dried over sodium sulfate,filtered, and concentrated under reduced pressure. The solid isrecrystallized with methanol.

Compound 314{8g}:2-(3-carboxymethylphenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(3-carboxymethylphenyl)-furan-3-one in dry THF underargon is added triethylamine in dry THF. The reaction is stirred at roomtemperature for 30 minutes, and 1 eq of phenylmethylsulfonyl chloride indry THF is added dropwise. The reaction is stirred for 16 hours followedby the addition of a saturated ammonium chloride. The mixture isextracted 3 times with 30 mL of diethyl ether. The combined etherextracts are washed with water and brine, dried over sodium sulfate,filtered, and concentrated under reduced pressure. The solid isrecrystallized with methanol.

Compound 315 {8h}:2-(3,4-dichlorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(3,4-dichlorophenyl)-furan-3-one in dry THF underargon is added triethylamine in dry THF. The reaction is stirred at roomtemperature for 30 minutes, and 1 eq of phenylmethylsulfonyl chloride indry THF is added dropwise. The reaction is stirred for 16 hours followedby the addition of a saturated ammonium chloride. The mixture isextracted 3 times with 30 mL of diethyl ether. The combined etherextracts are washed with water and brine, dried over sodium sulfate,filtered, and concentrated under reduced pressure. The solid isrecrystallized with methanol.

Compound 316 {8i}:2-(3,5-dichlorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(3,5-dichlorophenyl)-furan-3-one in dry THF underargon is added triethylamine in dry THF. The reaction is stirred at roomtemperature for 30 minutes, and 1 eq of phenylmethylsulfonyl chloride indry THF is added dropwise. The reaction is stirred for 16 hours followedby the addition of a saturated ammonium chloride solution. The mixtureis extracted 3 times with 30 mL of diethyl ether. The combined etherextracts are washed with water and brine, dried over sodium sulfate,filtered, and concentrated under reduced pressure. The solid isrecrystallized with methanol.

Compound 317 {8j}:2-(2-fluorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of 5-amino-4-hydroxy-2-(2-fluorophenyl)-furan-3-onein dry THF under argon is added triethylamine in dry THF. The reactionis stirred at room temperature for 30 minutes, and 1 eq ofphenylmethylsulfonyl chloride in dry THF is added dropwise. The reactionis stirred for 16 hours, followed by the addition of a saturatedammonium chloride solution. The mixture is extracted 3 times with 30 mLof diethyl ether. The combined ether extracts are washed with water andbrine, dried over sodium sulfate, filtered, and concentrated underreduced pressure. The solid is recrystallized with methanol.

Compound 318 {8k}:2-(3-fluorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of 5-amino-4-hydroxy-2-(3-fluorophenyl)-furan-3-onein dry THF under argon is added triethylamine in dry THF. The reactionis stirred at room temperature for 30 minutes, and 1 eq ofphenylmethylsulfonyl chloride in dry THF is added dropwise. The reactionis stirred for 16 hours followed by the addition of a saturated ammoniumchloride solution. The mixture is extracted 3 times with 30 mL ofdiethyl ether. The combined ether extracts are washed with water andbrine, dried over sodium sulfate, filtered, and concentrated underreduced pressure. The solid is recrystallized with methanol.

Compound 319 {8l}:2-(4-fluorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of 5-amino-4-hydroxy-2-(4-fluorophenyl)-furan-3-onein dry THF under argon is added triethylamine in dry THF. The reactionis stirred at room temperature for 30 minutes, and 1 eq ofphenylmethylsulfonyl chloride in dry THF is added dropwise. The reactionis stirred for 16 hours, followed by the addition of a saturatedammonium chloride solution. The mixture is extracted 3 times with 30 mLof diethyl ether. The combined ether extracts are washed with water andbrine, dried over sodium sulfate, filtered, and concentrated underreduced pressure. The solid is recrystallized with methanol.

Compound 320{8m}:2-(2,3-difluorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(2,3-difluorophenyl)-furan-3-one in dry THF underargon is added triethylamine in dry THF. The reaction is stirred at roomtemperature for 30 minutes, and 1 eq of phenylmethylsulfonyl chloride indry THF is added dropwise. The reaction is stirred for 16 hours,followed by the addition of a saturated ammonium chloride solution. Themixture is extracted 3 times with 30 mL of diethyl ether. The combinedether extracts are washed with water and brine, dried over sodiumsulfate, filtered, and concentrated under reduced pressure. The solid isrecrystallized with methanol.

Compound 321 {8n}:2-(2,4-difluorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(2,4-difluorophenyl)-furan-3-one in dry THF underargon is added triethylamine in dry THF. The reaction is stirred at roomtemperature for 30 minutes, and 1 eq of phenylmethylsulfonyl chloride indry THF is added dropwise. The reaction is stirred for 16 hours followedby the addition of a saturated ammonium chloride solution. The mixtureis extracted 3 times with 30 mL of diethyl ether. The combined etherextracts are washed with water and brine, dried over sodium sulfate,filtered, and concentrated under reduced pressure. The solid isrecrystallized with methanol.

Compound 322{8o}:2-(2,5-difluorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(2,5-difluorophenyl)-furan-3-one in dry THF underargon is added triethylamine in dry THF. The reaction is stirred at roomtemperature for 30 minutes, and 1 eq of phenylmethylsulfonyl chloride indry THF is added dropwise. The reaction is stirred for 16 hours followedby the addition of a saturated ammonium chloride solution. The mixtureis extracted 3 times with 30 mL of diethyl ether. The combined etherextracts are washed with water and brine, dried over sodium sulfate,filtered, and concentrated under reduced pressure. The solid isrecrystallized with methanol.

Compound 323{8p}:2-(2,6-difluorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(2,6-difluorophenyl)-furan-3-one in dry THF underargon is added triethylamine in dry THF. The reaction is stirred at roomtemperature for 30 minutes, and 1 eq of phenylmethylsulfonyl chloride indry THF is added dropwise. The reaction is stirred for 16 hours followedby the addition of a saturated ammonium chloride solution. The mixtureis extracted 3 times with 30 mL of diethyl ether. The combined etherextracts are washed with water and brine, dried over sodium sulfate,filtered, and concentrated under reduced pressure. The solid isrecrystallized with methanol.

Compound 324 {8q}:2-(3,4-difluorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(3,4-difluorophenyl)-furan-3-one in dry THF underargon is added triethylamine in dry THF. The reaction is stirred at roomtemperature for 30 minutes, and 1 eq of phenylmethylsulfonyl chloride indry THF is added dropwise. The reaction is stirred for 16 hours followedby the addition of a saturated ammonium chloride solution. The mixtureis extracted 3 times with 30 mL of diethyl ether. The combined etherextracts are washed with water and brine, dried over sodium sulfate,filtered, and concentrated under reduced pressure. The solid isrecrystallized with methanol.

Compound 325{8r}:2-(3,5-difluorophenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(3,5-difluorophenyl)-furan-3-one in dry THF underargon is added triethylamine in dry THF. The reaction is stirred at roomtemperature for 30 minutes, and 1 eq of phenylmethylsulfonyl chloride indry THF is added dropwise. The reaction is stirred for 16 hours followedby the addition of a saturated ammonium chloride solution. The mixtureis extracted 3 times with 30 mL of diethyl ether. The combined etherextracts are washed with water and brine, dried over sodium sulfate,filtered, and concentrated under reduced pressure. The solid isrecrystallized with methanol.

Compound 355 {8av}:2-(4-trifluoromethylphenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(4-trifluoromethylphenyl)-furan-3-one in dry THFunder argon is added triethylamine in dry THF. The reaction is stirringat room temperature for 30 minutes, and 1 eq of phenylmethylsulfonylchloride in dry THF is added dropwise. The reaction is stirred for 16hours followed by the addition of a saturated ammonium chloridesolution. The mixture is extracted 3 times with 30 mL of diethyl ether.The combined ether extracts are washed with water and brine, dried oversodium sulfate, filtered, and concentrated under reduced pressure. Thesolid is recrystallized with methanol.

Compound 358 {8ay}:2-(4-nitrilephenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(4-nitrilephenyl)-furan-3-one in dry THF under argonis added triethylamine in dry THF. The reaction is stirred at roomtemperature for 30 minutes, and 1 eq of phenylmethylsulfonyl chloride indry THF is added dropwise. The reaction is stirred for 16 hours followedby the addition of a saturated ammonium chloride solution. The mixtureis extracted 3 times with 30 mL of diethyl ether. The combined etherextracts are washed with water and brine, dried over sodium sulfate,filtered, and concentrated under reduced pressure. The solid isrecrystallized with methanol.

Compound 359{8az}:2-(3-nitrilephenyl)-4-[[phenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of5-amino-4-hydroxy-2-(3-nitrilephenyl)-furan-3-one in dry THF under argonis added triethylamine in dry THF. The reaction is stirred at roomtemperature for 30 minutes, and 1 eq of phenylmethylsulfonyl chloride indry THF is added dropwise. The reaction is stirred for 16 hours followedby the addition of a saturated ammonium chloride solution. The mixtureis extracted 3 times with 30 mL of diethyl ether. The combined etherextracts are washed with water and brine, dried over sodium sulfate,filtered, and concentrated under reduced pressure. The solid isrecrystallized with methanol.

Compound 368 {17c}:2-(4-chlorophenyl)-4-[[4-fluorophenylmethylsulfonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-onein dry THF under argon is added triethylamine in dry THF. The reactionis stirred at room temperature for 30 minutes, and 1 eq of4-fluorophenylmethylsulfonyl chloride in dry THF is added dropwise. Thereaction is stirred for 16 hours followed by the addition of a saturatedammonium chloride solution. The mixture is extracted 3 times with 30 mLof diethyl ether. The combined ether extracts are washed with water andbrine, dried over sodium sulfate, filtered, and concentrated underreduced pressure. The solid is recrystallized with methanol.

Compound 386{35c}:2-(4-chlorophenyl)-4-[[phenoxycarbonyl]oxy]-5-amino-3(2H)-furanone

To a solution of 5 g of 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-onein dry THF under argon is added triethylamine in dry THF. The reactionis stirred at room temperature for 30 minutes, and 1 eq ofphenylchloroformate in dry THF is added dropwise. The reaction isstirred for 16 hours followed by the addition of a saturated ammoniumchloride solution. The mixture is extracted 3 times with 30 mL ofdiethyl ether. The combined ether extracts are washed with water andbrine, dried over sodium sulfate, filtered, and concentrated underreduced pressure. The solid is recrystallized with methanol.

Compound 388 {37c}:2-(4-chlorophenyl)-4-[[phenylaminothiocarbonyl]oxy]-5-amino-3(2H)-furanone

To a stirring solution of 5 g of5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-one and sodium carbonate(1.7 g) in deionized water (30 mL) is added phenylisothiocyanate. Thereaction is stirred at room temperature for 24 hours. A saturatedammonium chloride solution is added and the mixture is extracted 3 timeswith 30 mL of diethyl ether. The combined ether extracts are washed withwater and brine, dried over sodium sulfate, filtered, and concentratedunder reduced pressure. The solid is recrystallized with methanol.

Example 2 Synthesis and Characterization of Compounds in Table 4 GeneralProcedures

Potassium cyanide (0.91 g) was added to sodium carbonate (1.7 g) indeionized water (30 mL) in a 3-Neck Glass Round Flask and placed in anice bath. The system was repeatedly purged using a vacuum pump andnitrogen gas. Glyoxal (3.72 g) was then added to the system without theintroduction of O₂ and the reactants were allowed to dissolve withstirring. In a stoppered tube, the appropriate arylaldehyde (7.11mmoles) was added to 1,4-dioxane (5 mL), purged, and then addeddrop-wise to the system. The system was then removed from the ice bathand allowed to stir at room temperature for 1 hour. After 1 hour, aceticacid (5 mL) was added drop-wise until gas bubbles were no longer visiblefrom the addition of acetic acid, or until the solution was at a pH ofless than 6. The solution was vacuum filtered and washed with ice coldwater (5 mL), methanol (5 mL) and ether (5 mL) and then was allowed toair dry. Crude material was recrystallized with methanol, collected byvacuum filtration and rinsed with diethyl ether and dried under vacuum.

Compound 401 {1c}: 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-one

Yield=70%. mp 221-2° C.; FTIR 3079, 1638; ¹H NMR (300 MHz, DMSO-d₆, ppm)δ 7.82 (s, 2H), 7.46 (d, J=8.7 Hz, 2H), 7.30 (s, 1H), 7.29 (d, J=8.7 Hz,2H), 5.43 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆, ppm) δ 182.6, 173.1, 135.7,133.2, 128.9, 128.6, 111.7, 82.2. HRMS Calc: 248.00849, Found: 248.00852MNa⁺═C₁₀H₈NO₃ClNa⁺; Elemental Analysis Calc: C 53.23, H 3.57, N 6.21, Cl15.71; Found: C 53.35, H 3.61, N 6.24, Cl 15.83 C₁₀H₈ClNO₃; HPLCretention time: 16.7 min.

Compound 455 {1j}: 5-amino-4-hydroxy-2-(2-fluorophenyl)-furan-3-one

Yield=84%. mp 160-3° C.; FTIR 3079, 1638; ¹H NMR (DMSO d₆) 5.60 (1H, s),7.22-7.78 (4H, m); HRMS Calc: 210.05610, Found: 210.05609 MH⁺=C₁₀H₉FNO₃⁺; Elemental Analysis Calc: C 57.42, H 3.85, N 6.70, F 9.08; Found: C57.50, H 3.92, N 6.61, F 8.99 C₁₀H₈FNO₃; HPLC retention time: 11.42 min.

Compound 456 {1k}: 5-amino-4-hydroxy-2-(3-fluorophenyl)-furan-3-one

Yield=60%. mp 168° C.; FTIR 3356, 3129; ¹H NMR (DMSO d₆) 5.44 (1H, s),7.05-7.27 (4H, m); HRMS Calc: 210.05610, Found: 210.05609 MH⁺=C₁₀H₉FNO₃+; Elemental Analysis Calc: C 57.42, H 3.85, N 6.70, F 9.08; Found: C57.41, H 3.87, N 6.61, F 8.97 C₁₀H₈FNO₃; HPLC retention time: 12.63 min.

Compound 457 {1l}: 5-amino-4-hydroxy-2-(4-fluorophenyl)-furan-3-one

Yield=68%. mp 160° C.; FTIR 3351, 3138; ¹H NMR (DMSO d₆) 5.41 (1H, s),7.22-7.78 (4H, m); HRMS Calc: 210.05610, Found: 210.05609 MH⁺=C₁₀H₉FNO₃⁺; Elemental Analysis Calc: C 57.42, H 3.85, N 6.70, F 9.08; Found: C57.42, H 3.97, N 6.66, F 8.90 C₁₀H₈FNO₃; HPLC retention time: 11.88 min.

Compound 458 {1m}: 5-amino-4-hydroxy-2-(2,3-difluorophenyl)-furan-3-one

Yield=76%. mp 193° C.; FTIR 3391, 3277, 1539; ¹H NMR (DMSO d₆) 5.68 (1H,s), 7.05-7.85 (3H, m); HRMS Calc: 228.04668, Found: 228.04669MH⁺=C₁₀H₈F₂NO₃ ⁺; Elemental Analysis Calc: C 52.87, H 3.11, N 6.17, F16.73; Found: C 53.10, H 3.11, N 6.17, F 16.57 C₁₀H₇F₂NO₃; HPLCretention time: 13.02 min.

Compound 459 {1n}: 5-amino-4-hydroxy-2-(2,4-difluorophenyl)-furan-3-one

Yield=67%. mp 182-3° C.; FTIR 3252, 1608; ¹H NMR (DMSO d₆) 5.59 (1H, s),7.13-7.79 (3H, m); HRMS Calc: 228.04668, Found: 228.04671 MH⁺=C₁₀H₈F₂NO₃⁺; Elemental Analysis Calc: C 52.87, H 3.11, N 6.17, F 16.73; Found: C52.84, H 3.00, N 6.16, F 16.59 C₁₀H₇F₂NO₃; HPLC retention time: 12.63min.

Compound 460 {1o}: 5-amino-4-hydroxy-2-(2,5-difluorophenyl)-furan-3-one

Yield=80%. mp 196° C.; FTIR 3391, 3267; ¹H NMR (DMSO d₆) 5.61 (1H, s),7.03-7.84 (3H, m); HRMS Calc: 228.04668, Found: 228.04670 MH⁺=C₁₀H₈F₂NO₃⁺; Elemental Analysis Calc: C 52.87, H 3.11, N 6.17, F 16.73; Found: C52.79, H 3.11, N 6.15, F 16.57 C₁₀H₇F₂NO₃; HPLC retention time: 12.09min.

Compound 461 {1p}: 5-amino-4-hydroxy-2-(2,6-difluorophenyl)-furan-3-one

Yield=70%. mp 159-60° C.; FTIR 3535, 3406; ¹H NMR (DMSO d₆) 5.68 (1H,s), 7.14-7.73 (3H, m); HRMS Calc: 228.04668, Found: 228.04670MH⁺=C₁₀H₈F₂NO₃ ⁺; Elemental Analysis Calc: C 52.87, H 3.11, N 6.17, F16.73; Found: C 52.62, H 3.10, N 5.94, F 16.57 C₁₀H₇F₂NO₃; HPLCretention time: 11.30 min.

Compound 462 {1q}: 5-amino-4-hydroxy-2-(3,4-difluorophenyl)-furan-3-one

Yield=76%. mp 190-4° C.; FTIR 3322, 3124; ¹H NMR (DMSO d₆) 5.44 (1H, s),7.13-7.85 (3H, m); HRMS Calc: 228.04668, Found: 228.04670 MH⁺=C₁₀H₈F₂NO₃ ⁺; Elemental Analysis Calc: C 52.87, H 3.11, N 6.17, F 16.73;Found: C 53.13, H 3.16, N 6.15, F 16.61 C₁₀H₇F₂NO₃; HPLC retention time:13.79 min.

Compound 463 {1r}: 5-amino-4-hydroxy-2-(3,5-difluorophenyl)-furan-3-one

Yield=58%. mp 190-1° C.; FTIR 3346, 3143; ¹H NMR (DMSO d₆) 5.68 (1H, s),7.05-7.85 (3H, m); HRMS Calc: 228.04668, Found: 228.04670 MH⁺=C₁₀H₈F₂NO₃⁺; Elemental Analysis Calc: C 52.87, H 3.11, N 6.17, F 16.73; Found: C52.88, H 3.06, N 6.15, F 16.70 C₁₀H₇F₂NO₃; HPLC retention time: 14.00min.

Compound 402 {40c or 2c}2-(4-chlorophenyl)-4-(acetoxy)-5-amino-3(2H)-furanone

1 g of 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-one was stirred inacetic anhydride under nitrogen gas for 16 hours. The reaction wascooled to −78° C., and lyophilized. The dried mass was recrystallizedfrom MeOH. Yield=44%. mp 221-2° C.; FTIR 3030, 1628; ¹H NMR (300 MHz,DMSO-d₆, ppm) δ 8.30 (s, 2H), 7.47 (d, J=8.7 Hz, 2H), 7.35 (d, J=8.7 Hz,2H), 5.63 (s, 1H), 2.14 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆, ppm) δ 182.4,173.0, 168.9, 134.8, 133.8, 129.1, 129.1, 106.9, 83.2, 20.7. ElementalAnalysis Calc: C 53.85, H 3.77, N 5.23; Found: C 53.76, H 3.90, N 5.24;C₁₂H₁₀ClNO₄; HPLC retention time: 19.7 min.

Compound 307 {3c REDUNDANT WITH 7C, IDENTICAL}2-(4-chlorophenyl)-4-[[phenylsulfonyl]oxy]-5-amino-3(2H)-furanone

5 g of 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-one was stirred in50 mL of dry THF under nitrogen gas for 16 hours with 4.7 g K₂CO₃ and4.25 mL of benzenesulfonyl chloride. The reaction was filtered, and thefiltrate was acidified with 24 mL 1N HCl, and extracted 5 times with 20mL of diethyl ether. The combined ether extracts were washed with brine,and dried with Na₂SO₄. After filtration, 100 mL of hexanes was added tothe solution, resulting in a precipitate, which was collected usingvacuum filtration and recrystallized with MeOH. Yield=17%. mp 190-195°C.; FTIR 3034, 1630; ¹H NMR (300 MHz, DMSO-d₆, ppm) δ 8.54 (s, 2H), 7.93(d, J=8.1 Hz, 2H), 7.74 (t, J=7.2 Hz, 1H), 7.57 (t, J=8.1 Hz, 2H), 7.48(d, J=8.7 Hz, 2H), 7.18 (d, J=8.4 Hz, 2H), 5.54 (s, 1H). ¹³C NMR (75MHz, DMSO-d₆, ppm) δ 181.2, 173.4, 135.2, 135.0, 134.2, 133.9, 129.7,129.2, 129.1, 129.0, 106.6, 82.9. Elemental Analysis Calc: C 52.54, H3.31, N 3.83, Cl 9.69; Found: C 52.50, H 3.33, N 3.79, Cl 9.84;C₁₆H₁₂ClNO₅S; HPLC retention time: 32.2 min.

Compound 404 {41c or 4c}:N-(3,4-dihydroxy-5-(4-chlorophenyl)-2-furanyl)methanesulfonamide

5 g of 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-one was stirred in50 mL of dry THF under nitrogen gas for 16 hours with 4.6 g K₂CO₃ and1.5 mL of methanesulfonyl chloride. The reaction was filtered, and thefiltrate was acidified with 24 mL 1N HCl, and extracted 5 times with 20mL of diethyl ether. The combined ether extracts were washed with brine,and dried with Na₂SO₄. After filtration, 100 mL of hexanes was added tothe solution, resulting in a precipitate, which was collected usingvacuum filtration and recrystallized with MeOH. The material was furtherpurified by column chromatography. Yield=18%. mp 175° C.; FTIR 3169,1616; ¹H NMR (300 MHz, DMSO-d₆, ppm) δ 8.41 (s, 1H), 8.09 (s, 1H), 7.96(d, J=8.4 Hz, 2H), 7.85 (s, 1H), 7.59 (d, J=8.4 Hz, 2H), 3.55 (s, 3H).¹³C NMR (75 MHz, DMSO-d₆, ppm) δ 185.7, 165.1, 140.7, 136.9, 135.9,133.5, 130.0, 129.7, 40.7. Elemental Analysis Calc: C 43.50, H 3.32, Cl11.67, N 4.61; Found: C 43.66, H 3.40, Cl 11.54, N 4.55; C₁₁H₁₀ClNO₅S;HPLC retention time: 25.2 min.

Compound 405 {42c or 5c}:N-(3,4-dihydroxy-5-(4-chlorophenyl)-2-furanyl)ethanesulfonamide

5 g of 5-amino-4-hydroxy-2-(4-chlorophenyl)-furan-3-one was stirred in50 mL of dry THF under nitrogen gas for 16 hours with 4.6 g K₂CO₃ and2.1 mL of ethanesulfonyl chloride. The reaction was filtered, and thefiltrate was acidified with 24 mL 1N HCl, and extracted 5 times with 20mL of diethyl ether. The combined ether extracts were washed with brine,and dried with Na₂SO₄. After filtration, 100 mL of hexanes was added tothe solution, resulting in a precipitate, which was collected usingvacuum filtration and recrystallized with ethyl acetate. Yield=21%. mp183-185° C.; FTIR 3181, 1616; ¹H NMR (300 MHz, DMSO-d₆, ppm) δ 8.41 (s,1H), 8.08 (s, 1H), 7.95 (d, J=9.3 Hz, 2H), 7.85 (s, 1H), 7.59 (d, J=9.0Hz, 2H), 3.68 (q, J=7.2 Hz, 2H), 1.40 (t, J=6.9 Hz, 3H). ¹³C NMR (75MHz, DMSO-d6, ppm) δ 185.8, 165.1, 140.7, 136.8, 136.1, 133.5, 130.0,129.7, 48.0, 8.6. Elemental Analysis Calc: C 45.36, H 3.81, Cl 11.16, N4.41; Found: C 45.42, H 3.85, Cl 11.06, N 4.37; C₁₂H₁₂ClNO₅S; HPLCretention time: 29.9 min.

Compound 406 {43c or 6c}:N-(3,4-dihydroxy-5-(4-chlorophenyl)-2-furanyl)benzenesulfonamide

5 g of 5-amino-4-hydroxy-2-(4-fluorophenyl)-furan-3-one is stirring in50 mL dry THF under dry nitrogen. The reaction is cooling in an icebath, and 1 equivalent of triethylamine is added dropwise. The reactionis warmed to room temperature, chlorotrimethylsilane is added dropwiseand the reaction is refluxing gently with a water bath for 30 minutes. 1equivalent of benzene sulfonyl chloride is added dropwise. 1 equivalentof TEA is added dropwise, and the reaction is gently refluxing with awater bath for 1 hour. The reaction is cooling to room temperature, and1 equivalent of tetrabutylammonium fluoride is added and stirs for 30minutes. A saturated ammonium sulfate solution is added to quench thereaction, and the reaction is extracted 3 times with 20 mL diethylether. The combined ether extracts are washed with water and brine, theether is dried with sodium sulfate, is filtered, and evaporates to yielda solid which is recrystallized with methanol.

Compound 407 {44c or 7c}:N-(3,4-dihydroxy-5-(4-chlorophenyl)-2-furanyl)acetamide

5 g of 5-amino-4-hydroxy-2-(4-fluorophenyl)-furan-3-one is stirring in50 mL dry THF under dry nitrogen. The reaction is cooling in an icebath, and 1 equivalent of triethylamine is added dropwise. The reactionis warmed to room temperature, chlorotrimethylsilane is added dropwiseand the reaction is refluxing gently with a water bath for 30 minutes. 1equivalent of acetyl chloride is added dropwise. 1 equivalent of TEA isadded dropwise, and the reaction is gently refluxing with a water bathfor 1 hour. The reaction is cooling to room temperature, and 1equivalent of tetrabutylammonium fluoride is added and stirs for 30minutes. A saturated ammonium sulfate solution is added to quench thereaction, and the reaction is extracted 3 times with 20 mL diethylether. The combined ether extracts are washed with water and brine, theether is dried with sodium sulfate, is filtered, and evaporates to yielda solid which is recrystallized with methanol.

Compound 409 {46c or 9c}:N-(3,4-dihydroxy-5-(4-chlorophenyl)-2-furanyl)benzamide

5 g of 5-amino-4-hydroxy-2-(4-fluorophenyl)-furan-3-one is stirring in50 mL dry THF under dry nitrogen. The reaction is cooling in an icebath, and 1 equivalent of triethylamine is added dropwise. The reactionis warmed to room temperature, chlorotrimethylsilane is added dropwiseand the reaction is refluxing gently with a water bath for 30 minutes. 1equivalent of benzoyl chloride is added dropwise. 1 equivalent of TEA isadded dropwise, and the reaction is gently refluxing with a water bathfor 1 hour. The reaction is cooling to room temperature, and 1equivalent of tetrabutylammonium fluoride is added and stirs for 30minutes. A saturated ammonium sulfate solution is added to quench thereaction, and the reaction is extracted 3 times with 20 mL diethylether. The combined ether extracts are washed with water and brine, theether is dried with sodium sulfate, is filtered, and evaporates to yielda solid which is recrystallized with methanol.

Compound 412 {48c or 12c}N-(3,4-dihydroxy-5-(4-chlorophenyl)-2-furanyl)succinimide

5 g of 5-amino-4-hydroxy-2-(4-fluorophenyl)-furan-3-one is stirring in50 mL pyridine under dry nitrogen. 1 equivalent of succinic anhydride isadded, and the reaction is refluxing gently with a water bath for 1hour. A saturated ammonium sulfate solution is added to quench thereaction, and the reaction is extracted 3 times with 20 mL diethylether. The combined ether extracts are washed with a saturatedbicarbonate solution and brine, the ether is dried with sodium sulfate,is filtered, and evaporates to yield a solid which is recrystallizedwith methanol.

TABLE 5Table of Nucleotide and Amino Acid Sequences Supported in the SpecificationSEQ ID Name Description Length Type NO EGF like domainDGDQCETSPC QNQGKCKDGL GEYTCTCLE 39 AA 1 peptide GFEGKNCELFEGF-like domain CDXXXCXXK XGNGXCDXXC NNAACXXDGX DC 31 AA 2 peptideconsensuscggaccgtgc aatggcccag cgtaagaatg ccaagagcag cggcaacagc agcagcagcg   612324 DNA 3 sequencegctccggcag cggtagcacg agtgcgggca gcagcagccc cggggcccgg agagagacaa  121cDNA sequenceagcatggagg acacaagaat gggaggaaag gcggactctc gggaacttca ttcttcacgt  181of humanggtttatggt gattgcattg ctgggcgtct ggacatctgt agctgtcgtt tggtttgatc  241ASPH (GENBANKttgttgacta tgaggaagtt ctaggaaaac taggaatcta tgatgctgat ggtgatggag  301Accessionattttgatgt ggatgatgcc aaagttttat taggacttaa agagagatct acttcagagc  361No. S83325;cagcagtccc gccagaagag gctgagccac acactgagcc cgaggagcag gttcctgtgg  421codon encodingaggcagaacc ccagaatatc gaagatgaag caaaagaaca aattcagtcc cttctccatg  481initiatingaaatggtaca cgcagaacat gttgagggag aagacttgca acaagaagat ggacccacag  541methionine isgagaaccaca acaagaggat gatgagtttc ttatggcgac tgatgtagat gatagatttg  601underlined)agaccctgga acctgaagta tctcatgaag aaaccgagca tagttaccac gtggaagaga  661cagtttcaca agactgtaat caggatatgg aagagatgat gtctgagcag gaaaatccag  721attccagtga accagtagta gaagatgaaa gattgcacca tgatacagat gatgtaacat  781accaagtcta tgaggaacaa gcagtatatg aacctctaga aaatgaaggg atagaaatca  841cagaagtaac tgctccccct gaggataatc ctgtagaaga ttcacaggta attgtagaag  901aagtaagcat ttttcctgtg gaagaacagc aggaagtacc accagaaaca aatagaaaaa  961cagatgatcc agaacaaaaa gcaaaagtta agaaaaagaa gcctaaactt ttaaataaat 1021ttgataagac tattaaagct gaacttgatg ctgcagaaaa actccgtaaa aggggaaaaa 1081ttgaggaagc agtgaatgca tttaaagaac tagtacgcaa ataccctcag agtccacgag 1141caagatatgg gaaggcgcag tgtgaggatg atttggctga gaagaggaga agtaatgagg 1201tgctacgtgg agccatcgag acctaccaag aggtggccag cctacctgat gtccctgcag 1261acctgctgaa gctgagtttg aagcgtcgct cagacaggca acaatttcta ggtcatatga 1321gaggttccct gcttaccctg cagagattag ttcaactatt tcccaatgat acttccttaa 1381aaaatgacct tggcgtggga tacctcttga taggagataa tgacaatgca aagaaagttt 1441atgaagaggt gctgagtgtg acacctaatg atggctttgc taaagtccat tatggcttca l501tcctgaaggc acagaacaaa attgctgaga gcatcccata tttaaaggaa ggaatagaat 1561ccggagatcc tggcactgat gatgggagat tttatttcca cctgggggat gccatgcaga 1621gggttgggaa caaagaggca tataagtggt atgagcttgg gcacaagaga ggacactttg 1681catctgtctg gcaacgctca ctctacaatg tgaatggact gaaagcacag ccttggtgga 1741ccccaaaaga aacgggctac acagagttag taaagtcttt agaaagaaac tggaagttaa 1801tccgagatga aggccttgca gtgatggata aagccaaagg tctcttcctg cctgaggatg 1861aaaacctgag ggaaaaaggg gactggagcc agttcacgct gtggcagcaa ggaagaagaa 1921atgaaaatgc ctgcaaagga gctcctaaaa cctgtacctt actagaaaag ttccccgaga 1981caacaggatg cagaagagga cagatcaaat attccatcat gcaccccggg actcacgtgt 2041ggccgcacac agggcccaca aactgcaggc tccgaatgca cctgggcttg gtgattccca 2101aggaaggctg caagattcga tgtgccaacg agaccaggac ctgggaggaa ggcaaggtgc 2161tcatctttga tgactccttt gagcacgagg tatggcagga tgcctcatct ttccggctga 2221tattcatcgt ggatgtgtgg catccggaac tgacaccaca gcagagacgc agccttccag 2281caatttagca tgaattcatg caagcttggg aaactctgga gaga Amino acidMAQRKNAKSS GNSSSSGSGS GSTSAGSSSP GARRETKHGG HKNGRKGGLS GTSFFTWFMV   61758 AA 4 sequence ofIALLGVWTSV AVVWFDLVDY EEVLGKLGIY DADGDGDFDV DDAKVLLGLK ERSTSEPAVP  121human ASPH PEEAEPHTEP EEQVPVEAEP QNIEDEAKEQ IQSLLHEMVH AEHVEGEDLQ QEDGPTGEPQ  181(GenBankQEDDEFLMAT DVDDRFETLE PEVSHEETEH SYHVEETVSQ DCNQDMEEMM SEQENPDSSE  241Accession No.PVVEDERLHH DTDDVTYQVY EEQAVYEPLE NEGIEITEVT APPEDNPVED SQVIVEEVSI  301S83325; HisFPVEEQQEVP PETNRKTDDP EQKAKVKKKK PKLLNKFDKT IKAELDAAEK LRKRGKIEEA  361motif isVNAFKELVRK YPQSPRARYG KAQCEDDLAE KRRSNEVLRG AIETYQEVAS LPDVPADLLK  421underlined;LSLKRRSDRQ QFLGHMRGSL LTLQRLVQLF PNDTSLKNDL GVGYLLIGDN DNAKKVYEEV  481conservedLSVTPNDGFA KVHYGFILKA QNKIAESIPY LKEGIESGDP GTDDGRFYFH LGDAMQRVGN  541sequencesKEAYKWYELG HKRGHFASVW QRSLYNVNGL KAQPWWTPKE TGYTELVKSL ERNWKLIRDE  601withinGLAVMDKAKG LFLPEDENLR EKGDWSQFTL WQQGRRNENA CKGAPKTCTL LEKFPETTGC  661the catalyticRRGQIKYSIM HPGTHVWPHT GPTNCRLRMH LGLVIPKEGC KIRCANETRT WEEGKVLIFD  721domain are DSFEHEVWQD ASSFRLIFIV DVWHPELTPQ QRRSLPAI designated bybold type)

Other Embodiments

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

REFERENCES

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

Patent Documents

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Journal Articles

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1. An asparatyl (asparaginyl) beta-hydroxylase (ASPH) inhibitorycompound for use in a method of reducing proliferation, migration,invasion, or metastasis of a tumor cell in the treatment of cellproliferative disorder, comprising contacting said tumor cell with theASPH inhibitory compound, wherein the ASPH inhibitory compound is ofFormula Ia or Ib:

or a salt, ester, metabolite, prodrug, or solvate thereof, wherein Ar¹is substituted or unsubstituted C₆-C₂₀ aryl or 5 to 20-memberedheteroaryl; X is C(O), C(S), or S(O)₂; W¹ is a single bond, O, CR⁵⁰R⁵¹,or NR⁵² when X is CO and W¹ is a single bond, CR⁵⁰R⁵¹, or NR⁵² when X isSO₂; and each of R⁵⁰, R⁵¹, R⁵², and R⁵³ independently is selected fromthe group consisting of hydrogen, substituted or unsubstituted C₁-C₆alkyl, substituted or unsubstituted C₂-C₆ alkenyl, substituted orunsubstituted C₂-C₆ alkynyl, substituted or unsubstituted C₆-C₂₀ aryl,substituted or unsubstituted C₇-C₂₆ arylalkyl, substituted orunsubstituted 5 to 20-membered heteroaryl, and substituted orunsubstituted 6-26 membered heteroarylalkyl.
 2. The ASPH inhibitorycompound for use of claim 1, wherein the compound is of Formula Ia, or asalt, ester, metabolite, prodrug, or solvate thereof.
 3. The ASPHinhibitory compound for use of claim 2, wherein the compound is ofFormula IIa:

or a salt, ester, metabolite, prodrug, or solvate thereof, wherein eachof Ar¹ and Ar² independently is unsubstituted C₆-C₁₄ aryl, unsubstituted5 to 14-membered heteroaryl, or C₆-C₁₄ aryl or 5 to 14-memberedheteroaryl each substituted with one or more substituents selected fromthe group consisting of halo, CN, NO₂, NO, N₃, OR_(a), NR_(a)R_(b),C(O)R_(a), C(O)OR_(a), C(O)NR_(a)R_(b), NR_(b)C(O)R_(a), —S(O)_(b)R_(a),—S(O)_(b)NR_(a)R_(b), or R_(S1), in which R_(S1) is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5- or 6-memberedheteroaryl, or 4 to 12-membered heterocycloalkyl, b is 0, 1, or 2, eachof R_(a) and R_(b), independently is H or R_(S2), and R_(S2) is C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl; and eachof R_(S1) and R_(S2), is optionally substituted with one or moresubstituents selected from the group consisting of halo, OH, oxo,C(O)OH, C(O)O—C₁-C₆ alkyl, CN, C₁-C₆ alkyl, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6-memberedheteroaryl.
 4. The ASPH inhibitory compound for use of claim 2, whereinR⁵³ is unsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substituted with one ormore substituents selected from halo, OH, CN, and amino.
 5. The ASPHinhibitory compound for use of claim 2, wherein X is S(O)₂ and W¹ isCR⁵⁰R⁵¹.
 6. The ASPH inhibitory compound for use of claim 2, wherein Xis S(O)₂ and W¹ is a single bond.
 7. The ASPH inhibitory compound foruse of claim 2, wherein X is C(O) and W¹ is O, or X is C(S) and W¹ isNR⁵².
 8. The ASPH inhibitory compound for use of claim 2, wherein eachof R⁵⁰, R⁵¹, and R⁵² independently is H, unsubstituted C₁-C₆ alkyl, orC₁-C₆ alkyl substituted with one or more substituents selected fromhalo, OH, CN, and amino.
 9. The ASPH inhibitory compound for use ofclaim 2, wherein each of Ar¹ and Ar² independently is phenyl, naphthyl,or 5 to 10-membered heteroaryl, each of which is optionally substitutedwith one or more substituents selected from the group consisting ofhalo, CN, NO₂, NO, N₃, OR_(a), NR_(a)R_(b), C(O)R_(a), C(O)OR_(a),C(O)NR_(a)R_(b), NR_(b)C(O)R_(a), —S(O)_(b)R_(a), —S(O)_(b)NR_(a)R_(b),or R_(S1), in which R_(S1) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5- or 6-membered heteroaryl, or 4 to12-membered heterocycloalkyl, b is 0, 1, or 2, each of R_(a) and R_(b),independently is H or R_(S2), and R_(S2) is C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, or 5- or 6-membered heteroaryl; and each of R_(S1) andR_(S2), is optionally substituted with one or more substituents selectedfrom the group consisting of halo, OH, oxo, C(O)OH, C(O)O—C₁-C₆ alkyl,CN, C₁-C₆ alkyl, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl.
 10. The ASPHinhibitory compound for use of claim 9, wherein each of Ar¹ and Ar²independently is phenyl, naphthyl, or 5 to 10-membered heteroaryl, eachof which is optionally substituted with one or more substituentsselected from the group consisting of halo, CN, NO₂, NO, N₃, OR_(a),NR_(a)R_(b), C(O)R_(a), C(O)OR_(a), or RS1, in which R_(S1) is C₁-C₆alkyl, each of R_(a) and R_(b), independently is H or R_(S2), and R_(S2)is C₁-C₆ alkyl; and each of R_(S1) and R_(S2), is optionally substitutedwith one or more substituents selected from the group consisting ofhalo, OH, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, and di-C₁-C₆alkylamino.
 11. The ASPH inhibitory compound for use of claim 9, whereineach of Ar¹ and Ar² independently is selected from phenyl, 1-naphthyl,2-naphthyl, 2-furanyl, 2-thiazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,2-quinolinyl, 3-quinolinyl, 4-quinolinyl, 2-chlorophenyl,3-chlorophenyl, 4-chlorophenyl, 2-fluorophenyl, 3-fluorophenyl,4-fluorophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl,4-trifluoromethylphenyl, 2-cyanophenyl, 3-cyanophenyl, 4-cyanophenyl,3-carboxymethylphenyl, 2-methoxyphenyl, 3-methoxyphenyl,4-methoxyphenyl, 2,3-dichlorophenyl, 2,4-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl,3,4-difluorophenyl, 3,5-difluorophenyl, 2,3-dimethoxyphenyl,2,4-dimethoxyphenyl, 2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl,3,5-dimethoxyphenyl, 2-chloro-6-fluorophenyl, 3-chloro-4-fluorophenyl,2-chloro-4-fluorophenyl, 4-chloro-3-fluorophenyl,3-chloro-2-fluorophenyl, 2-chloro-5-fluorophenyl,4-chloro-2-fluorophenyl, and 5-chloro-2-fluorophenyl.
 12. The ASPHinhibitory compound for use of claim 1, wherein the compound is ofFormula Ib, or a salt, ester, metabolite, prodrug, or solvate thereof.13. The ASPH inhibitory compound for use of claim 12, wherein R⁵³ isunsubstituted C₁-C₆ alkyl or C₁-C₆ alkyl substituted with one or moresubstituents selected from halo, OH, CN, and amino.
 14. The ASPHinhibitory compound for use of claim 12, wherein R⁵³ is unsubstitutedmethyl or ethyl.
 15. The ASPH inhibitory compound for use of claim 12,wherein X is S(O)₂ and W¹ is CR⁵⁰R⁵¹.
 16. The ASPH inhibitory compoundfor use of claim 12, wherein X is S(O)₂ and W¹ is a single bond.
 17. TheASPH inhibitory compound for use of claim 12, wherein X is C(O) and W¹is O, or X is C(S) and W¹ is NR⁵².
 18. The ASPH inhibitory compound foruse of claim 12, wherein each of R⁵⁰, R⁵¹, and R⁵² independently is H,unsubstituted C₁-C₆ alkyl, or C₁-C₆ alkyl substituted with one or moresubstituents selected from halo, OH, CN, and amino.
 19. The ASPHinhibitory compound for use of claim 12, wherein Ar¹ is phenyl,naphthyl, or 5 to 10-membered heteroaryl, each of which is optionallysubstituted with one or more substituents selected from the groupconsisting of halo, CN, NO₂, NO, N₃, OR_(a), NR_(a)R_(b), C(O)R_(a),C(O)OR_(a), C(O)NR_(a)R_(b), NR_(b)C(O)R_(a), —S(O)_(b)R_(a),—S(O)_(b)NR_(a)R_(b), or R_(S1), in which R_(S1) is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5- or 6-memberedheteroaryl, or 4 to 12-membered heterocycloalkyl, b is 0, 1, or 2, eachof R_(a) and R_(b), independently is H or R_(S2), and R_(S2) is C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl; and eachof R_(S1) and R_(S2), is optionally substituted with one or moresubstituents selected from the group consisting of halo, OH, oxo,C(O)OH, C(O)O—C₁-C₆ alkyl, CN, C₁-C₆ alkyl, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6-memberedheteroaryl.
 20. The ASPH inhibitory compound for use of claim 19,wherein Ar¹ is phenyl, naphthyl, or 5 to 10-membered heteroaryl, each ofwhich is optionally substituted with one or more substituents selectedfrom the group consisting of halo, CN, NO₂, NO, N₃, OR_(a), NR_(a)R_(b),C(O)R_(a), C(O)OR_(a), or R_(S1), in which R_(S1) is C₁-C₆ alkyl, eachof R_(a) and R_(b), independently is H or R_(S2), and R_(S2) is C₁-C₆alkyl; and each of R_(S1) and R_(S2), is optionally substituted with oneor more substituents selected from the group consisting of halo, OH,C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, and di-C₁-C₆ alkylamino.21. The ASPH inhibitory compound for use of claim 19, wherein Ar¹ isselected from phenyl, 1-naphthyl, 2-naphthyl, 2-furanyl, 2-thiazolyl,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-quinolinyl, 3-quinolinyl,4-quinolinyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl,2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2-trifluoromethylphenyl,3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-cyanophenyl,3-cyanophenyl, 4-cyanophenyl, 3-carboxymethylphenyl, 2-methoxyphenyl,3-methoxyphenyl, 4-methoxyphenyl, 2,3-dichlorophenyl,2,4-dichlorophenyl, 2,5-dichlorophenyl, 3,4-dichlorophenyl,3,5-dichlorophenyl, 2,3-difluorophenyl, 2,4-difluorophenyl,2,5-difluorophenyl, 3 ,4-difluorophenyl, 3 ,5-difluorophenyl,2,3-dimethoxyphenyl, 2,4-dimethoxyphenyl, 2,5-dimethoxyphenyl,3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 2-chloro-6-fluorophenyl,3-chloro-4-fluorophenyl, 2-chloro-4-fluorophenyl,4-chloro-3-fluorophenyl, 3-chloro-2-fluorophenyl,2-chloro-5-fluorophenyl, 4-chloro-2-fluorophenyl, and5-chloro-2-fluorophenyl.
 22. The ASPH inhibitory compound for use ofclaim 1, wherein the compound is selected from the compounds listed inTables 3 and
 4. 23. The ASPH inhibitory compound for use of claim 1,wherein said tumor cell expresses ASPH.
 24. The ASPH inhibitory compoundfor use of claim 1, wherein said cell proliferative disorder comprisesPancreatic Cancer, Hepatocellular Cancer, Cholangiocarcinoma, LungCancer, Colon Cancer, Breast Cancer, Prostatic Cancer, and Glioblastoma.25. The ASPH inhibitory compound for use of claim 1, wherein saidcompound is administered intravenously, orally, or subcutaneously. 26.The ASPH inhibitory compound for use of claim 1, wherein said compoundis administered at a dose of 0.01 to 50 milligrams/kilogram of bodyweight.
 27. A compound of Formula Ia:

or a salt, ester, metabolite, prodrug, or solvate thereof, wherein Ar¹is substituted or unsubstituted C₆-C₂₀ aryl or 5 to 20-memberedheteroaryl; X is C(O), C(S), or S(O)₂; W¹ is a single bond, O, CR⁵⁰R⁵¹,or NR⁵² when X is CO and W¹ is a single bond, CR⁵⁰R⁵¹, or NR⁵² when X isSO₂; and each of R⁵⁰, R⁵¹, R⁵², and R⁵³ independently is selected fromthe group consisting of hydrogen, substituted or unsubstituted C₁-C₆alkyl, substituted or unsubstituted C₂-C₆ alkenyl, substituted orunsubstituted C₂-C₆ alkynyl, substituted or unsubstituted C₆-C₂₀ aryl,substituted or unsubstituted C₇-C₂₆ arylalkyl, substituted orunsubstituted 5 to 20-membered heteroaryl, and substituted orunsubstituted 6-26 membered heteroarylalkyl, provided that when Ar¹ is4-chlorophenyl, then R⁵³ is not methyl or unsubstituted phenyl.
 28. Thecompound of claim 27, the compound is of Formula IIa:

or a salt, ester, metabolite, prodrug, or solvate thereof, wherein eachof Ar¹ and Ar² independently is unsubstituted C₆-C₁₄ aryl, unsubstituted5 to 14-membered heteroaryl, or C₆-C₁₄ aryl or 5 to 14-memberedheteroaryl each substituted with one or more substituents selected fromthe group consisting of halo, CN, NO₂, NO, N₃, OR_(a), NR_(a)R_(b),C(O)R_(a), C(O)OR_(a), C(O)NR_(a)R_(b), NR_(b)C(O)R_(a), —S(O)_(b)R_(a),—S(O)_(b)NR_(a)R_(b), or R_(S1), in which R_(S1) is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5- or 6-memberedheteroaryl, or 4 to 12-membered heterocycloalkyl, b is 0, 1, or 2, eachof R_(a) and R_(b), independently is H or R_(S2), and R_(S2) is C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl; and eachof R_(S1) and R_(S2), is optionally substituted with one or moresubstituents selected from the group consisting of halo, OH, oxo,C(O)OH, C(O)O—C₁-C₆ alkyl, CN, C₁-C₆ alkyl, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6-memberedheteroaryl.
 29. The compound of claim 27, wherein R⁵³ is unsubstitutedC₁-C₆ alkyl or C₁-C₆ alkyl substituted with one or more substituentsselected from halo, OH, CN, and amino.
 30. The compound of claim 27,wherein X is S(O)₂ and W¹ is CR⁵⁰R⁵¹.
 31. The compound of claim 27,wherein X is S(O)₂ and W¹ is a single bond.
 32. The compound of claim27, wherein X is C(O) and W¹ is O, or X is C(S) and W¹ is NR⁵².
 33. Thecompound of claim 27, wherein each of R⁵⁰, R⁵¹, and R⁵² independently isH, unsubstituted C₁-C₆ alkyl, or C₁-C₆ alkyl substituted with one ormore substituents selected from halo, OH, CN, and amino.
 34. Thecompound of claim 27, wherein each of Ar¹ and Ar² independently isphenyl, naphthyl, or 5 to 10-membered heteroaryl, each of which isoptionally substituted with one or more substituents selected from thegroup consisting of halo, CN, NO₂, NO, N₃, OR_(a), NR_(a)R_(b),C(O)R_(a), C(O)OR_(a), C(O)NR_(a)R_(b), NR_(b)C(O)R_(a), —S(O)_(b)R_(a),—S(O)_(b)NR_(a)R_(b), or R_(S1), in which R_(S1) is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5- or 6-memberedheteroaryl, or 4 to 12-membered heterocycloalkyl, b is 0, 1, or 2, eachof R_(a) and R_(b), independently is H or R_(S2), and R_(S2) is C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl; and eachof R_(S1) and R_(S2), is optionally substituted with one or moresubstituents selected from the group consisting of halo, OH, oxo,C(O)OH, C(O)O—C₁-C₆ alkyl, CN, C₁-C₆ alkyl, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6-memberedheteroaryl.
 35. The compound of claim 34, wherein each of Ar¹ and Ar²independently is phenyl, naphthyl, or 5 to 10-membered heteroaryl, eachof which is optionally substituted with one or more substituentsselected from the group consisting of halo, CN, NO₂, NO, N₃, OR_(a),NR_(a)R_(b), C(O)R_(a), C(O)OR_(a), or R_(S1), in which R_(S1) is C₁-C₆alkyl, each of R_(a) and R_(b), independently is H or R_(S2), and R_(S2)is C₁-C₆ alkyl; and each of R_(S1) and R_(S2), is optionally substitutedwith one or more substituents selected from the group consisting ofhalo, OH, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, and di-C₁-C₆alkylamino.
 36. The compound of claim 34, wherein each of Ar¹ and Ar²independently is selected from phenyl, 1-naphthyl, 2-naphthyl,2-furanyl, 2-thiazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-quinolinyl,3-quinolinyl, 4-quinolinyl, 2-chlorophenyl, 3-chlorophenyl,4-chlorophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl,2-trifluoromethylphenyl, 3-trifluoromethylphenyl,4-trifluoromethylphenyl, 2-cyanophenyl, 3-cyanophenyl, 4-cyanophenyl,3-carboxymethylphenyl, 2-methoxyphenyl, 3-methoxyphenyl,4-methoxyphenyl, 2,3-dichlorophenyl, 2,4-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl, 3,4-difluorophenyl, 3 ,5-difluorophenyl, 2,3-dimethoxyphenyl,2,4-dimethoxyphenyl, 2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl,3,5-dimethoxyphenyl, 2-chloro-6-fluorophenyl, 3-chloro-4-fluorophenyl,2-chloro-4-fluorophenyl, 4-chloro-3-fluorophenyl,3-chloro-2-fluorophenyl, 2-chloro-5-fluorophenyl,4-chloro-2-fluorophenyl, and 5-chloro-2-fluorophenyl.
 37. The compoundof claim 27, wherein the compound is selected from Table
 3. 38. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of claim 27 or a salt, ester, metabolite,prodrug, or solvate thereof.
 39. A method of producing a compound ofclaim 30, comprising contacting an amine compound of Formula (IIIa)

with a sulfonyl chloride of the formula ClSO₂(CR⁵⁰R⁵¹)Ar² under asuitable condition to produce a compound of claim
 30. 40. A method fortreating or preventing a cell proliferative disorder, comprisingadministering to a subject in need thereof a therapeutically efficientamount of a compound of Formula Ia or Ib:

or a salt, ester, metabolite, prodrug, or solvate thereof, wherein Ar¹is substituted or unsubstituted C₆-C₂₀ aryl or 5 to 20-memberedheteroaryl; X is C(O), C(S), or S(O)₂; W¹ is a single bond, O, CR⁵⁰R⁵¹,or NR⁵² when X is CO and W¹ is a single bond, CR⁵⁰R⁵¹, or NR⁵² when X isSO₂; and each of R⁵⁰, R⁵¹, R⁵², and R⁵³ independently is selected fromthe group consisting of hydrogen, substituted or unsubstituted C₁-C₆alkyl, substituted or unsubstituted C₂-C₆ alkenyl, substituted orunsubstituted C₂-C₆ alkynyl, substituted or unsubstituted C₆-C₂₀ aryl,substituted or unsubstituted C₇-C₂₆ arylalkyl, substituted orunsubstituted 5 to 20-membered heteroaryl, and substituted orunsubstituted 6-26 membered heteroarylalkyl.
 41. The method of claim 40,wherein the compound is of Formula IIa:

or a salt, ester, metabolite, prodrug, or solvate thereof, wherein eachof Ar¹ and Ar² independently is unsubstituted C₆-C₁₄ aryl, unsubstituted5 to 14-membered heteroaryl, or C₆-C₁₄ aryl or 5 to 14-memberedheteroaryl each substituted with one or more substituents selected fromthe group consisting of halo, CN, NO₂, NO, N₃, OR_(a), NR_(a)R_(b),C(O)R_(a), C(O)OR_(a), C(O)NR_(a)R_(b), NR_(b)C(O)R_(a), —S(O)_(b)R_(a),—S(O)_(b)NR_(a)R_(b), or R_(S1), in which R_(S1) is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 5- or 6-memberedheteroaryl, or 4 to 12-membered heterocycloalkyl, b is 0, 1, or 2, eachof R_(a) and R_(b), independently is H or R_(S2), and R_(S2) is C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl; and eachof R_(S1) and R_(S2), is optionally substituted with one or moresubstituents selected from the group consisting of halo, OH, oxo,C(O)OH, C(O)O—C₁-C₆ alkyl, CN, C₁-C₆ alkyl, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6-memberedheteroaryl.
 42. The method of claim 40, wherein the compound is ofFormula Ib, or a salt, ester, metabolite, prodrug, or solvate thereof.43. The method of claim 42, wherein R⁵³ is unsubstituted C₁-C₆ alkyl orC₁-C₆ alkyl substituted with one or more substituents selected fromhalo, OH, CN, and amino.
 44. A method determining ASPH activity,contacting an EGF-like domain peptide with a detectably-labeledα-ketoglutarate and ASPH enzyme and measuring β-hydroxylase activity.45. The method of claim 44, wherein said α-ketoglutarate is¹⁴C-labelled, and wherein β-hydroxylase activity is measured bydetecting liberated ¹⁴CO₂.
 46. The method of claim 45, wherein saidliberated ¹⁴CO₂ is captured on a filter and radioactivity quantified.47. The method of claim 44, further comprising contacting said ASPHenzyme with a candidate compound, wherein a decrease in β-hydroxylaseactivity in the presence of said compound compared to the absence ofsaid compound indicates that said compound inhibits ASPH enzymeactivity.
 48. The method of claim 44, wherein said EGF-like domaincomprises the amino acid sequence of (SEQ ID NO: 1)DGDQCETSPCQNQGKCKDGLGEYTCTCLEGFEGKNCELF .


49. The method of claim 44, wherein said EGF-like domain peptidecomprises the consensus sequence of (SEQ ID NO: 2)CDXXXCXXKXGNGXCDXXCNNAACXXDGXDC .